Reconciling genealogical and morphological species in a worldwide study of the Family Hydractiniidae...

© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta,

38

, 4, July 2009, pp403–430

403

Miglietta, M. P., Schuchert, P. & Cunningham, C. W. (2009). Reconciling genealogical andmorphological species in a worldwide study of the Family Hydractiniidae (Cnidaria, Hydrozoa).—

Zoologica Scripta

,

38

, 403–430.The Hydractiniidae are a family of globally distributed marine hydrozoans (class Hydrozoa,phylum Cnidaria). Despite being one of the most well-studied families of the Hydrozoa, theirgenus and species-level taxonomy is unsettled and disputed. The taxonomic difficulties of theHydractiniidae are due to many inadequate species descriptions, a paucity of availablemorphological characters, many cryptic species, and the often-extreme plasticity seen whencolonies of the same species are found at different stages of growth or different environmentalconditions. This confusion over species identity is especially important because some speciesof the family Hydractiniidae are well-established model organisms for a wide array of studiesranging from gene expression to developmental biology and colony growth. Here we reportthe species-level implications of 226 mitochondrial large ribosomal subunit (16S) rDNAsequences from around the world and 52 nuclear DNA sequences (Elongation Factor 1

!

) withthe intent to reconcile described morphospecies with genealogical lineages.Our data show that

Podocoryna carnea

and

P. exigua

are distinct and geographically disjunctspecies,

P. borealis

is paraphyletic with respect to

Podocoryna

sp. from South Africa and

P. bella

from New Zealand.

Podocoryna australis,

from New Zealand form a distinct monophyleticgroup.

Podocoryna

from New England, New York and Florida all fall into a distinct monophyleticgroup (

P. americana

) and fail to support the existence of a distinct,

P. selena

in the Gulf ofMexico.

Hydractinia pruvoti

is the only species within the

Podocoryna

clade without fully formedmedusae. We identify a

Clava

clade closely related to other algae dwelling Hydractiniidae. Ourdata do not recover

Stylactaria inabai

from Japan as a distinct species from

S. misakiensis

, and

S. carcinicola

as distinct from

H. epiconcha

. Also, 10 colonies identified as

S. carcinicola

fall intoa distantly related clade that is close to the American

S. hooperi.

Finally, we identify

Janariamirabilis

as the sister group to the

H. echinata

species complex and clarify the relationshipsbetween the

H. echinata

,

H. symbiopollicaris

,

H. polyclina

,

H. symbiolongicarpus

and

H.

[GM].Corresponding author:

Maria Pia Miglietta, Biology Department, Pennsylvania State University,University Park, PA 16802, USA. E-mail: [email protected]

Current address:

Biology Department, Pennsylvania State University, University Park, PA 16802, USAPeter Schuchert, Museum d’Histoire Naturelle, CH1211, Route malagnou 1, Geneva, Switzerland.E-mail: [email protected] Cunningham, Department of Biology, Duke University, Durham, NC 27708, USA. E-mail:[email protected]

Blackwell Publishing Ltd

Reconciling genealogical and morphological species in a worldwide study of the Family Hydractiniidae (Cnidaria, Hydrozoa)

M

ARIA

P

IA

M

IGLIETTA

,* P

ETER

S

CHUCHERT

& C

LIFFORD

W. C

UNNINGHAM

Submitted: 7 July 2008Accepted: 10 November 2008doi:10.1111/j.1463-6409.2008.00376.x

Introduction

The Hydractiniidae are a family of globally distributedmarine hydrozoans (class Hydrozoa, phylum Cnidaria). Thebasic hydrozoan life cycle includes a benthic colonial stage thatreproduces asexually, and a pelagic sexual medusa (jellyfish)stage that is asexually produced by the colony (Fig. 1). Whilesome hydractiniid species retain the complete polyp/medusalife cycle, in most hydractiniid species the swimming medusa

stage is reduced to the point where it is never released fromthe colony. The polyps of hydrozoan colonies are connectedto one another by gastrovascular canals known as stolons(Fig. 1). Hydractiniid colonies typically grow on marine surfacesas epibionts, with most living on gastropod shells occupied byliving gastropods or by hermit crabs (Mills 1976; Buss &Yund 1989; Schuchert 2008). Other hydractiniid coloniesgrow on algae or rock substrata. The family Hydractiniidae

Reconciling genealogical and morphological species in the Hydractiniidae

•

M. P. Miglietta

et al.

404

Zoologica Scripta,

38

, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters

comprises about 106 nominal species and 10 extant genera(Bouillon

et al

. 2006).Despite being one of the most well-studied families of the

Hydrozoa, the genus and species-level taxonomy is unsettledand disputed (Schuchert 2008; Boero

et al

. 1998). The taxonomicdifficulties of the Hydractiniidae are due both to a paucity ofmorphological characters, and the often-extreme plasticityseen when colonies of the same species are found at differentstages of growth or different environmental conditions. Herewe report the species-level implications of 226 mitochondriallarge ribosomal subunit (16S) rDNA sequences from aroundthe world.

Difficulty of diagnosing hydractiniid genera

The three main and most speciose genera of the Hydractiniidae(

Hydractinia

van Beneden 1867;

Podocoryna

Sars 1846; and

Stylactaria

Stechow 1921a) are traditionally distinguishedwith a combination of two morphological and developmentalcharacters, one involving the developmental stage of sexuallymature medusae, and the other involving the developmentalstage of the colony itself. The likelihood that the major generaare not monophyletic led Bouillon

et al

. (1997), Boero

et al

.(1998) and Bouillon

et al

. (2006) to propose merging

Hydractinia, Stylactaria

and

Podocoryna

into the oldest one —

Hydractinia.

These authors used the argument that neitherthe state of the hydrorhiza nor the presence/absence of amedusa allows an unambiguous separation of the genera due

to intergrading forms. The present paper does not aim toresolve the genus-level discussion; this will be done in aseparate study that includes more nuclear genes. Some genusallocations used in this article are thus provisional andcorrespond more to common usage than strict monophyleticclades.

Degree of medusa reduction in hydractiniid genera

The genus

Podocoryna

is characterized by having fully-formedswimming medusae that are asexually produced from thebenthic colony stage. These medusae are normally fullyformed, that is, they have radial canals, tentacles and a manu-brium with a functional mouth. In all the other hydractiniidgenera — including the largest genera (

Stylactaria

and

Hydractinia

) — the medusae becomes sexually mature beforethey complete development (paedomorphosis, Boero & Sara1987; Cunningham & Buss 1993). There are two levels ofpaedomorphosis in the Hydractiniidae, eumedusoids andsporosacs. Eumedusoids are partially formed medusae thatbecome sexually mature without completing development tothe point where they can swim normally. Depending on thespecies or the environmental conditions, eumedusoids aresometimes released from the colony, but cannot move farbefore they release their gametes. In other cases, eumedusoidsare never released from the colony, effectively ending thealternation of asexual and sexual generations that characterizethe Hydrozoa. Sporosacs represent the most extreme case of

Fig. 1 A, B. General hydrozoan life cycle with fully functional medusa (typical of the genus Podocoryna, Fam. Hydractiniidae) (A) and withmedusa reduced to sporosac (B). Intermediate forms, with medusae reduced to non-feeding, swimming medusoids are also found in theHydrozoa in general and in the Family Hydractiniidae.

M. P. Miglietta

et al.

•

Reconciling genealogical and morphological species in the Hydractiniidae

© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta,

38

, 4, July 2009, pp403–430

405

paedomorphosis, so that gametes are produced inside sacsthat are not only attached to the colony, but have lost anytraces of the medusa stage, most notably the radial canals thatare present in eumedusoids.

Characteristics of colony formation in hydractiniid genera

The genera

Stylactaria

and

Hydractinia

have been diagnosedby the structure of the hydrorhiza, the network of stolonsconnecting the polyps (Millard 1975; Calder 1988; Hirohito1988; Schuchert 1996). The hydrorhiza of

Stylactaria

is formedby stolons — often forming a close reticulated meshwork —that are always enclosed in the non-living, chitinous periderm.The hydrorhiza of

Hydractinia

colonies is composed ofstolons that coalesce into a mat whose top layer is composedof living tissue, formally referred to as naked coenosarc.Chitin is produced in other parts of

Hydractinia

colonies, butnot on the top layer of the mat. In many species of the genus

Podocoryna

— which differs from the other genera by producingfree-swimming fully-formed medusae — colonies routinelyprogress from periderm-covered reticulate stolons to amore fully-developed coalescence of stolons with nakedcoenosarc. In terms of timing of development, these can bedescribed as events of heterochrony (Blackstone 1996). The

Stylactaria

condition can be described as paedomorphic,since it stops at the periderm-covered stage, and the

Hydractinia

condition can be described as accelerated since itreaches the naked coenosarc stage well before achievingreproductive maturity (Blackstone 1996).

Rare hydractiniid genera

Other genera in the Hydractiniidae are

Hydrissa

Stechow1921

, Clavactinia

Thornely 1904,

Janaria

Stechow 1921a,

Hydrocorella

Stechow 1921b,

Fiordlandia

Schuchert 1996,

Kinetocodium

Kramp 1922

, Hansiella

Bouillon 1980, and

Tregubovia

Picard 1958 (see Bouillon

et al

. 2006). Thesegenera are generally rare, and morphologically very distinct.For example,

Janaria

and

Hydrocorella

form fully calcifiedskeletons. Finally, the genus

Clava

was originally placed inthe Family Clavidae, but has been recently reassigned to theHydractiniidae (Schuchert 2001).

Difficulty of diagnosing hydractiniid species

Species-level identification is famously difficult not only inthe Hydractiniidae but in the entire class Hydrozoa. Many ofthe more than 100 nominal species of Hydractiniidae are atpresent not recognizable due to inadequate descriptions (seealso Schuchert 2008). Many species are known only from theoriginal — and sometimes very old — descriptions, some datingback more than a century and often lacking morphologicalinformation now considered important for correct identification.

Adding to the confusion, these animals show extrememorphological plasticity depending on the substratum or

environmental conditions. As described below, morphotypesof the same species have been described as different species,resulting in an overestimation of species diversity. On theother hand, the presence of sibling species with little or nomorphological diversification leads to an underestimation ofthis diversity. As an example, the most thoroughly studiedspecies of the family has usually been referred to in the literature— regardless of where it was collected — as

Hydractinia echinata

(Fleming 1828). This was divided into four sibling species,with

H. echinata

in Europe, and three morphologicallyvery similar species in North America (

H. symbiolongicarpus,H. symbiopollicaris

and

H. polyclina

Buss & Yund 1989). Eventhis did not reflect the extent of the cryptic diversity. Asdescribed below, there is a fourth, undescribed

Hydractinia

species in the Gulf of Mexico, and the European

Hydractiniaechinata

is itself composed of two distinct species.This confusion over species identity is especially important

because some species of the family Hydractiniidae are well-established model organisms for a wide array of studiesranging from gene expression to developmental biology andcolony growth (Yund

et al

. 1987; Schierwater

et al

. 1991;Blackstone & Buss 1993; Blackstone 1996; Cartwright

et al

.1999). Given the lack of clarity and necessary details of somespecies description, together with the intrinsic difficulty totell Hydractiniidae species apart, many experimental studiesmight have been carried out on material of dubious identity(Boero

et al

. 1998).

An mtDNA-based analysis of worldwide collection of hydractiniid species

This article investigates mitochondrial and nuclear genegenealogies for 226 Hydractiniidae colonies or medusaecollected worldwide (Table 1). We report an effort to reconciledescribed morphospecies with monophyletic groups recon-structed using fragments of the mitochondrial large ribosomalsubunit gene (16S) and the nuclear gene Elongation Factor1

!

. The 16S gene is much more easily amplified in Hydrozoathan COI. Although the 16S gene evolves about one-third asfast as COI (Govindarajan

et al

. 2005), comparisons of severalcryptic species in the hydrozoan genus

Obelia

did not find anycases where a monophyletic group was identified by COI butnot 16S (Govindarajan

et al

. 2005), and the same is true forseveral hydractiniid species (Cunningham, unpublishedobservations). Where possible, we apply a genealogicalspecies concept to provisionally identify evolutionarily signifi-cant units (Baum & Shaw 1995).

Materials and methods

Collection of fresh material

For a complete list of the 226 samples, localities, voucherspecimens, and GenBank accession numbers see Table 1.Sequences belonging to seven species of the family Stylasteridae

Reconciling genealogical and m

orphological species in the Hydractiniidae

•

M. P. M

iglietta

et al.

406

Zoologica Scripta,

38

, 4, July 2009, pp403–430•

© 2009 The Authors. Journal com

pilation © 2009 The Norw

egian Academy of Science and Letters

Table 1

List of the vaucher specimens, primary species identification in the filed, collecting sites, substrates, GenBank accession numbers, and identifier.

Specimen code in trees Primary species identification Collecting sites Susbstate Voucher specimen Identified byGenBank accession number 16S/EF1

!

1 045

Clavactina gallensis

Clavactinia gallensis

Hua Hin, Gulf of Siam,Thailand Gastropod MHNG INVE33470 P. Schuchert FJ2143772 046

H. serrata

USA

Hydractinia serrata

Friday Harbor, WA, USA Hermit crab — C. Cunningham xxx–xxx3 047

H. rubricata

NZ

Hydractinia rubricata

New Zealand Hermit crab paratypes P. Schuchert FJ2143784 48b

H. serrata

USA

Hydractinia serrata

Friday Harbor, WA, USA Hermit crab — C. Cunningham xxx-xxx5 050

H. serrata

USA

Hydractinia serrata

Bering Sea, AK, USA Hermit crab — C. Cunningham FJ2145946 052

H. serrata

USA

Hydractinia serrata

Bering Sea, AK, USA Hermit crab — C. Cunningham FJ2145957 053

H. serrata

USA

Hydractinia serrata

Friday Harbor, WA, USA Hemit crab — C. Cunningham FJ2145978 012

Hydractinia

spec.

Hydractinia

spec. 2 provenience unknown — — A. Collins FJ2143799 072

H. polyclina

USA

Hydractinia polyclina

Woods Hole, MA, USA

Pagurus acadianus

— L. Buss Lab. xxx–xxx/FJ37285610 070

H. echinata Hydractinia echinata

Roscoff, France Hermit crab — L. Buss Lab. xxx–xxx/FJ37285511 071

H. echinata

USA

Hydractinia echinata

New York Harbor, NY, USA Hermit crab — M.P. Miglietta FJ21455612 074


USA

Hydractinia symbiolongicarpus

Woods Hole, MA, USA

Pagurus longicarpus

— M.P. Miglietta FJ21438013 073


USA


New York Harbor, NY, USA

Pagurus longicarpus

— M.P. Miglietta FJ21455214 075


USA


Beaufort, NC, USA Pagurus longicarpus — L. Buss Lab. FJ214551/FJ37285715 065 Hydrissa sodalis Japan Hydrissa sodalis Okushiri Is., Hokkaido, Japan Hermit crab — M.P. Miglietta FJ21454716 066 Hydrissa sodalis Japan Hydrissa sodalis Okushiri Is., Hokkaido, Japan Hermit crab — M.P. Miglietta FJ21454817 067 Hydrissa sodalis Japan Hydrissa sodalis Okushiri Is., Hokkaido, Japan Hermit crab — M.P. Miglietta FJ21455318 068 Hydrissa sodalis Japan Hydrissa sodalis Okushiri Is., Hokkaido, Japan Hermit crab — M.P. Miglietta FJ214554/FJ37285319 069 Janaria mirabilis Janaria mirabilis Gulf of Mexico Hermit crab — L. Buss Lab FJ214555/FJ37285420 077 H. altispina S. Africa Hydractinia altispina False Bay, South Africa Gastropod — M.P. Miglietta FJ214381/FJ37285821 078 Hydractinia sp. California Hydractinia sp. Monterey Bay, CA, USA Sea weed (Codium) — A. Govindarajan FJ214382/FJ37285922 079 S uchidai Japan Stylactaria uchidai Muroran, Pacific coast of Hokkaido, Japan Hermit crab — L. Buss Lab. FJ21438323 081 H. laevispina Hydractinia laevispina Catalina Island, CA, USA — — L. Buss Lab. FJ21438624 080 H. milleri Canada Hydractinia milleri Vancouver, Canada Dock Piles — A. Brinkman-Voss FJ214385/FJ37286025 082 H. milleri USA Hydractinia milleri Bodega Bay, CA, USA Rock — L. Buss Lab. FJ214384/FJ37289926 083 S. reticulata Japan Stylactaria reticulata Choshi, Boso Peninsula, Japan — — H. Namikawa FJ214387/FJ37286127 084 H. epiconcha Japan Hydractinia epiconcha Kominato, Boso Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21438828 085 H. epiconcha Japan Hydractinia epiconcha Hirido beach, Nakagi, Izu Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21438929 086 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21439030 087 S. carcinicola Japan Stylactaria carcinicola Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Turbo sp. — M.P.Miglietta FJ21439131 088 S. carcinicola Japan Stylactaria carcinicola Misaki, Sagami Bay, Miura Peninsula, Japan Turbo sp. — M.P. Miglietta FJ214392/FJ37286232 089 S. carcinicola Japan Stylactaria carcinicola Misaki Fish Market, Sagami Bay, Japan Turbo sp. — M.P. Miglietta FJ21439333 090 H. epiconcha Japan Hydractinia epiconcha Kominato, Boso Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21439434 091 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Turbo sp. — M.P. Miglietta FJ21439535 092 S. carcinicola Japan Stylactaria carcinicola Hirido beach, Nakagi, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21439636 093 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21439737 094 H. epiconcha Japan Hydractinia epiconcha Kominato, Boso Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21439838 095 H. epiconcha Japan Hydractinia epiconcha Kominato, Boso Peninsula, Japan Gastropod — M.P. Miglietta FJ21439939 096 H. epiconcha Japan Hydractinia epiconcha Hirido beach, Nakagi, Izu Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21440040 097 H. epiconcha Japan Hydractinia epiconcha Misaki Fish Market, Sagami Bay, Japan Turbo sp. — M.P. Miglietta FJ21440141 098 S. carcinicola Japan Stylactaria carcinicola Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Gastropod — M.P. Miglietta FJ21440242 099 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ214403

M. P. M

iglietta et al.•






•Zoologica Scripta, 38, 4, July 2009, pp403–430

407

43 100 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan Gastropod – M.P. Miglietta FJ21440444 101 H. epiconcha Japan Hydractinia epiconcha Shimoda Bay, Izu Peninsula, Japan Bufoniella sp. – M.P. Miglietta FJ21440545 102 H. epiconcha Japan Hydractinia epiconcha Hirido beach, Nakagi, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21440646 051 H. epiconcha Japan Hydractinia epiconcha Kominato, Boso Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21440747 104 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Hermit crab — M.P. Miglietta FJ21440848 105 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ214409/FJ37286349 106 S. carcinicola Japan Stylactaria carcinicola Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Turbo sp. — M.P. Miglietta FJ21441050 107 S. carcinicola Japan Stylactaria carcinicola Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Turbo sp. — M.P. Miglietta FJ21441151 108 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan — — M.P. Miglietta FJ21441252 109 H. epiconcha Japan Hydractinia epiconcha Hirido beach, Nakagi, Izu Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21441353 110 S. carcinicola Japan Stylactaria carcinicola Mkimoto Pearl Island, Toba, Kii Peninsula, Japan Turbo sp. — M.P. Miglietta FJ21441454 111 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21441555 112 H. epiconcha Japan Hydractinia epiconcha Shimoda Bay, Izu Peninsula, Japan Pollia mollis — M.P. Miglietta FJ214416/FJ37286456 114 H. epiconcha Japan Hydractinia epiconcha Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21441757 115 H. epiconcha Japan Hydractinia epiconcha Hirido beach, Nakagi, Izu Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21441858 116 H. epiconcha Japan Hydractinia epiconcha Shimoda Bay, Izu Peninsula, Japan Pollia mollis — M.P. Miglietta FJ21441959 117 H. epiconcha Japan Hydractinia epiconcha Kominato, Boso Peninsula, Japan Gastropod — M.P. Miglietta FJ21442060 118 H. epiconcha Japan Hydractinia epiconcha Kominato, Boso Peninsula, Japan Pollia mollis — M.P. Miglietta FJ214421/FJ37286561 119 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21442262 120 H. epiconcha Japan Hydractinia epiconcha Japan Gastropod — M.P. Miglietta FJ21442363 121 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan Turbo sp. — M.P. Miglietta FJ21442464 122 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ21442565 123 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21442666 124 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21442767 125 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ21442868 126 S. carcinicola Japan Stylactaria carcinicola Ito, Izu Peninsula, Japan Hermit crab — M.P. Miglietta FJ21442969 127 H. allmani Iceland Hydractinia allmanii Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ214430/FJ37286670 128 Hydractinia sp. USA Hydractinia sp. unidentified CA, USA Rock — A. Lindner FJ214431/FJ37286771 129 H. antonii Aleutians Hydractinia antonii Aleutian Islands, AK, USA Rock — NOAA cruise 2000 FJ21443272 130 H. allmanii Bering Sea Hydractinia allmanii Bering Sea, AK, USA Hermit crab — M.P. Miglietta FJ214433/FJ37286873 131 S. conchicola Japan Stylactaria conchicola Oshoro, Japan Sea coast of Hokkaido, Japan Homalopoma amussitatum — M.P. Miglietta FJ21443474 132 S. conchicola Japan Stylactaria conchicola Oshoro, Japan Sea coast of Hokkaido, Japan Homalopoma amussitatum — M.P. Miglietta FJ214435/FJ37286975 133 S. conchicola Japan Stylactaria conchicola Oshoro, Japan Sea coast of Hokkaido, Japan Homalopoma amussitatum — M.P. Miglietta FJ21443676 134 H. fucicola Italy Hydractinia fucicola Torre del Serpe, Otranto, Apulia, Italy Sea weed — S. Piraino FJ214437/FJ37287077 001 H. inermis Italy Hydractinia inermis Otranto, Apulia, Italy Sea weed — M.P. Miglietta FJ21450278 002 H. inermis Italy Hydractinia inermis Otranto, Apulia, Italy Sea weed — M.P. Miglietta FJ21454479 007 H. inermis Italy Hydractinia inermis Otranto, Apulia, Italy Sea weed — M.P. Miglietta FJ21454580 008 H. inermis Italy Hydractinia inermis Otranto, Apulia, Italy Sea weed — M.P. Miglietta FJ21454681 135 Clava multicornis Iceland Clava multicornis Sangerdi vicinity, Iceland Sea weed — M.P. Miglietta FJ21443882 136 Clava multicornis Iceland Clava multicornis Sangerdi vicinity, Iceland Sea weed — M.P. Miglietta FJ2144383 137 Clava multicornis USA Clava multicornis Woods Hole, MA, USA Sea weed — M.P. Miglietta FJ21444084 138 Clava multicornis USA Clava multicornis Woods Hole, MA, USA Sea weed — M.P. Miglietta FJ21444185 139 Clava multicornis USA Clava multicornis Woods Hole, MA, USA Sea weed — L. Buss Lab. FJ214442

Specimen code in trees Primary species identification Collecting sites Susbstate Voucher specimen Identified byGenBank accession number 16S/EF1!

Table 1 Continued.



•M

. P. Miglietta et al.

408Zoologica Scripta, 38, 4, July 2009, pp403–430

•©

2009 The Authors. Journal compilation ©

2009 The Norwegian Academ

y of Science and Letters

86 140 Clava multicornis USA Clava multicornis NY, USA Sea weed — M.P. Miglietta FJ214443

87 141 Clava multicornis Europe Clava multicornis Europe Sea weed — L. Buss Lab. FJ21444488 142 P. americana USA Podocoryna americana Woods Hole, MA, USA Hermit crab — M.P. Miglietta FJ21444589 143 P. americana USA Podocoryna americana Long Island, USA Hermit crab — L. Buss Lab. FJ21444690 144 P. americana USA Podocoryna americana Woods Hole, MA, USA Hermit crab — M.P. Miglietta FJ21444891 145 P. americana USA Podocoryna americana Woods Hole, MA, USA Hermit crab — L. Buss Lab. FJ21444792 146 P. americana USA Podocoryna americana Woods Hole, MA, USA Hermit crab — M.P. Miglietta FJ21444993 147 P. americana USA Podocoryna americana Woods Hole, MA, USA Pagurus pollicaris — M.P. Miglietta FJ214450/FJ37290394 148 P. americana USA Podocoryna americana Woods Hole, MA, USA Pagurus longicarpus — M.P. Miglietta FJ214451/FJ37290495 149 P. borealis Iceland Podocoryna borealis Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ21445296 150 P. borealis Iceland Podocoryna borealis Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ21445397 151 P. borealis Iceland Podocoryna borealis Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ214454/FJ37287198 152 P. borealis Iceland Podocoryna borealis Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ21445599 153 P. borealis Iceland Podocoryna borealis Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ214456100 154 P. borealis Iceland Podocoryna borealis Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ214457101 155 P. borealis Iceland Podocoryna borealis Keijflavic Harbour, Iceland Hermit crab — M.P. Miglietta FJ214458102 156 P. borealis Podocoryna borealis Keijflavic Harbour, Iceland — — M.P. Miglietta FJ214459/FJ372872103 157 P. borealis Iceland Podocoryna borealis Aquarium, Sangerdi Marine Lab., Iceland Rock — M.P. Miglietta FJ214460104 158 P. borealis Scotland Podocoryna borealis Dunstaffnage, Scotland *Medusae Identical to AY787878 P. Schuchert FJ214461105 159 P. bella New Zealand Podocoryna bella Otago, New Zealand Fish — M.P. Miglietta FJ214462/FJ372873106 160 Podocoryna spec. Podocoryna spec. SA Kalk Bay, South Africa *Medusae — M.P. Miglietta FJ214463/FJ372874107 161 P. borealis Iceland Podocoryna borealis Aquarium, Sangerdi Marine Lab., Iceland Shell — M.P. Miglietta FJ214464108 162 P. borealis Iceland Podocoryna borealis Keflavic Harbour, Iceland Gastropod — M.P. Miglietta FJ214465109 163 P. australis New Zealand Podocoryna australis Otago, New Zealand — — M.P. Miglietta FJ214466/FJ372875110 164 P. australis New Zealand Podocoryna australis New Zealand — material Schuchert (1996) P. Schuchert FJ214467111 165 P. australis New Zealand Podocoryna australis New Zealand — material Schuchert (1996) P.Schuchert FJ214468/FJ372876112 166 P. carnea Denmark Podocoryna carnea Denmark — — L. Buss Lab. FJ214469/FJ372877113 167 P. exigua France Podocoryna exigua Banyuls, France Hinia incrassata — P. Schuchert FJ214470114 168 P. exigua France Podocoryna exigua Banyuls, France Murex brandaris — P. Schuchert FJ214471115 169 P. exigua France Podocoryna exigua Banyuls, France Hinia incrassata — P. Schuchert FJ214472116 170 P. exigua France Podocoryna exigua Banyuls, France Hinia incrassata — P. Schuchert FJ214473117 171 P. exigua France Podocoryna exigua Roscoff, France — — L. Buss Lab. FJ214474118 172 P. exigua France Podocoryna exigua Mediterranean, France — — L. Buss Lab. FJ214475119 173 P. exigua Italy Podocoryna exigua Otranto, Apulia, Italy — — F. Boero FJ214476/FJ372878120 174 P. exigua France Podocoryna exigua Banyuls, France Murex brandaris — P. Schuchert FJ214477121 175 P. exigua France Podocoryna exigua Banyuls, France Murex brandaris — P. Schuchert FJ214478122 176 P. exigua France Podocoryna exigua Banyuls, France Hinia incrassata — P. Schuchert FJ214479123 177 P. exigua France Podocoryna exigua Banyuls, France Hinia incrassata — P. Schuchert FJ214480124 178 P. exigua France Podocoryna exigua Banyuls, France Barnacles — P. Schuchert FJ214481125 179 P. exigua France Podocoryna exigua Banyuls, France Hinia incrassata — P. Schuchert FJ214482126 180 P. exigua France Podocoryna exigua Banyuls, France Hinia incrassata — P. Schuchert FJ214483127 P. selena P. selena Florida — — C. Cunningham xxx—xxx


Table 1 Continued.

M. P. M

iglietta et al.•







409

128 181 P. exigua France Podocoryna exigua Banyuls, France Hermit crab — P. Schuchert FJ214484129 182 H. pruvoti France Hydractinia pruvoti Banyuls, France Hermit crab MHNG INVE32973 P. Schuchert FJ214485/FJ372879130 183 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Crab — M.P. Miglietta FJ214486

131 184 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Hermit crab — M.P. Miglietta FJ214487132 185 P. hayamaensis Japan Podocoryna hayamaensis Shimoda Bay, Izu Peninsula, Japan Crab — M.P. Miglietta FJ214488133 186 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Hermit crab — M.P. Miglietta FJ214489134 187 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Crab — M.P. Miglietta FJ214490135 188 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Crab — M.P. Miglietta FJ214491136 189 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Hermit crab — M.P. Miglietta FJ214492/FJ372880137 190 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Crab — M.P. Miglietta FJ214493/FJ372902138 005 Stylactaria sp. Japan Stylactaria sp. 1 on Sargassum Kominato, Boso Peninsula, Japan Sargassum sp. — Yakko Hirano FJ214494139 192 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Hermit crab — M.P. Miglietta FJ214495140 193 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Hermit crab — M.P. Miglietta FJ214496141 194 P. hayamaensis Japan Podocoryna hayamaensis Ushimado, Seto Inland Sea, Japan Hermit crab — M.P. Miglietta FJ214497/FJ372901142 196 Hydractinia sp. Ushimado Hydractina sp. Ushimado, Seto Inland Sea, Japan Gastropod — M.P. Miglietta FJ214498/FJ372882143 197 S. hooperi California Stylactaria hooperi sp. 2 Monterey Bay, CA, USA Gastropod — L. Buss Lab. FJ214499/FJ372881144 198 S. carcinicola Japan Stylactaria carcinicola Hirido beach, Nakagi, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ214500145 199 S. carcinicola Japan Stylactaria carcinicola Kominato, Boso Peninsula, Japan Hermit crab — M.P. Miglietta FJ214501/FJ372883146 200 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan Rope (in aquarium) — M.P. Miglietta FJ214503/FJ372884147 201 S. carcinicola Japan Ito Stylactaria carcinicola Ito, Izu Peninsula, Japan Crab — M.P. Miglietta FJ214504148 202 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan Crab — M.P. Miglietta FJ214505149 203 Hydractina cf. calderi Italy Hydractina cf. calderi Otranto, Apulia, Italy Gastropod — M.P. Miglietta FJ214506150 204 S. carcinicola Japan Stylactaria carcinicola Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214507/FJ372885151 205 Stylactaria hooperi USA Stylactaria hooperi sp. 1 Woods Hole, MA, USA Dead shell — M.P. Miglietta FJ214508/FJ372886152 206 S. carcinicola Japan Stylactaria carcinicola sp. 3 Ito, Izu Peninsula, Japan Hermit crab — M.P. Miglietta FJ214509153 207 Hydractina cf. calderi Italy Hydractina cf. calderi Otranto, Apulia, Italy Gastropod — M.P. Miglietta FJ214510/FJ372887154 208 S. carcinicola Japan Stylactaria carcinicola sp. 3 Ito, Izu Peninsula, Japan — — M.P. Miglietta FJ214511155 209 S. carcinicola Japan Stylactaria carcinicola sp. 3 Loc. unknown, Japan Crab — M.P. Miglietta FJ214512156 210 S. carcinicola Japan Stylactaria carcinicola sp. 3 Mikimoto Pearl Island, Toba, Kii Peninsula, Japan Turbo sp. — M.P. Miglietta FJ214513157 211 S. misakiensis Ushimado Stylactaria misakiensis Nishiwaki beach, Ushimado, Seto Inland Sea, Japan Gastropod — M.P. Miglietta FJ214514/FJ372888158 212 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Gastropod — M.P. Miglietta FJ214515159 213 S. misakiensis Japan Stylactaria misakiensis Kashino, Ushimado, Seto Inland Sea, Japan Gastropod — M.P. Miglietta FJ214516160 214 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214517161 215 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214518162 216 S. misakiensis Japan Stylactaria misakiensis Nishiwaki beach, Ushimado, Seto Inland Sea, Japan Gastropod — M.P. Miglietta FJ214519/FJ372889163 217 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Nassarius multigranosus — M.P. Miglietta FJ214520/FJ372890164 218 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Nassarius multigranosus — M.P. Miglietta FJ214521/FJ372891165 219 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Nassarius multigranosus — M.P. Miglietta FJ214522166 220 S. misakiensis Japan Stylactaris misakiensis Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214523167 221 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Nassarius multigranosus — M.P. Miglietta FJ214524/FJ372892168 222 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214525169 223 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214526/FJ372893170 225 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Nassarius multigranosus — M.P. Miglietta FJ214528


Table 1 Continued.



•M



•©




171 224 S. inabai Japan Stylactaria inabai Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214527/FJ372894172 226 S inabai Japan Stylactaria inabai Shimoda Bay, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ214529173 227 S. inabai Japan Stylactaria inabai Misaki, Sagami Bay, Miura Peninsula, Japan Gastropod — M.P. Miglietta FJ214530174 228 S inabai Japan Stylactaria inabai Shimoda Bay, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ214531/FJ372895175 229 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ214532176 230 S. sp. Japan Stylactaria inabai Nabeta, Shimoda Bay, Japan Hermit crab — M.P. Miglietta FJ214533177 231 S. inabai Japan Stylactaria inabai Misaki, Sagami Bay, Miura Peninsula, Japan Crab — M.P. Miglietta FJ214534178 232 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214535179 233 S. inabai Japan Stylactaria inabai Misaki, Sagami Bay, Miura Peninsula, Japan Gastropod — M.P. Miglietta FJ214536180 234 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan — — M.P. Miglietta FJ214537/FJ372896181 235 S. misakiensis Japan Stylactaria misakiensis Shimoda Bay, Izu Peninsula, Japan Gastropod — M.P. Miglietta FJ214538182 237 S. inabai Japan Stylactaria inabai Shimoda Bay, Izu Peninsula, Japan Hermit crab — M.P. Miglietta FJ214539183 238 S. misakiensis Japan Stylactaria misakiensis Kashino, Ushimado, Seto Island Sea, Japan Gastropod — M.P. Miglietta FJ214540184 239 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Nassarius multigranosus — M.P. Miglietta FJ214541185 240 S. sp. Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan Nassarius multigranosus — M.P. Miglietta FJ214542/FJ372898186 241 S. multigranosi Japan Stylactaria multigranosi Oshoro, Japan Sea coast of Hokkaido, Japan, Nassarius multigranosus — M.P. Miglietta FJ214543187 071 H. echinata Hydractinia echinata Scotland Hermit crab — L.A. Henry xxx—xxx188 029 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214557189 026 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214558190 043 Hydractinia Gulf of Mexico Hydractinia new spec. Panacea, Gulf of Mexico, FL, USA Hermit crab — C. Cunningham FJ214559191 044 Hydractinia Gulf of Mexico Hydractinia new spec. Panacea, Gurf of Mexico, FL, USA Hermit crab — C. Cunningham FJ214560192 009 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214561193 030 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214592194 172 H. echinata Denmark Hydractinia echinata Fredrikshavn, Denmark Hermit crab — C. Cunningham FJ214562195 173 H. echinata Denmark Hydractinia echinata Fredrikshavn, Denmark Hermit crab — C. Cunningham FJ214563196 045 Hydractinia Gulf of Mexico Hydractinia new spec. Panacea, Gurf of Mexico, FL, USA Hermit crab — C. Cunningham FJ214564197 012 H. echinata Belgium Hydractinia echinata Belgium Hermit crab — C. Cunningham FJ214565198 011 H. echinata Belgium Hydractinia echinata Belgium Hermit crab — C. Cunningham FJ214566199 010 H. echinata Belgium Hydractinia echinata Belgium Hermit crab — C. Cunningham FJ214567200 017 H. echinata Belgium Hydractinia echinata Belgium Hermit crab — C. Cunningham FJ214568201 015 H. echinata Belgium Hydractinia echinata Belgium Hermit crab — C. Cunningham FJ214569202 038 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214570203 038 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham xxx—xxx204 040 H. symbiolongicarpus USA Hydractinia symbiolongicarpus Long Island, USA Pagurus longicarpus — C. Cunningham FJ214571205 041 H. symbiolongicarpus USA Hydractinia symbiolongicarpus Long Island, USA Pagurus longicarpus — C. Cunningham FJ214572206 042 H. symbiolongicarpus USA Hydractinia symbiolongicarpus Long Island, USA Pagurus longicarpus — C. Cunningham FJ214573207 020 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214574208 021 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214575209 033 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214576210 013 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214577211 034 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214578212 032 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214579/FJ372900


Table 1 Continued.

M. P. M

iglietta et al.•







411

213 028 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214580214 034 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham xxx—xxx215 025 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214581216 039 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214582217 036 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214583218 019 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214584219 024 H. symbiopollicaris USA Hydractinia symbiopollicaris Woods Hole, MA, USA Pagurus pollicaris — C. Cunningham FJ214585220 027 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214586221 023 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214587222 035 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214588223 014 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214589224 018 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214590225 016 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214591226 030 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214592227 037 H. polyclina USA Hydractinia polyclina Maine, USA Pagurus acadianus — C. Cunningham FJ214593228 053 Adelopora crassilabrum Adelopora crassilabrum Aramis Seamount, Norfolk Ridge, New Caledonia Rock USNM1027760 A. Lindner xxx—xxx229 052 Stylaster duchassaingi Stylaster duchassaingi Bahamas Rock — A. Lindner xxx—xxx230 060 Stylaster roseus Stylaster roseus Grenada, Spain Rock USNM1078387 A. Lindner xxx—xxx231 061 Stylasteridae spec. Stylasteriidae unidet. Aleutian Islands, AK, USA Rock — A. Lindner xxx—xxx232 059 Stylaster sanguineus Stylaster sanguineus Palau Rock — A. Lindner xxx—xxx233 054 Errinopora nannecea Errinopora nanneca Aleutian Islands, AK, USA Rock USNM1027820 A. Lindner xxx—xxx234 063 Stylaster sp. Stylantheca petrograpta Race Rocks, British Columbia, Canada Rock — A. Lindner xxx—xxx


Table 1 Continued.

Reconciling genealogical and morphological species in the Hydractiniidae • M. P. Miglietta et al.

412 Zoologica Scripta, 38, 4, July 2009, pp403–430 • © 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters

were made available by A. Lindner. Samples will be depositedat the Smithsonian Institution National Museum of NaturalHistory (NMNH, Washington, DC, USA). Some specimensare also described by Schuchert (2008) and have been depositedin the Natural History Museum of Geneva (MHNG), andothers were previously deposited in the Peabody Museumat Yale University (see Appendix I for specimen voucherinformation).

Colonies were collected by SCUBA diving, snorkelling,trawling and dredging on various habitats, and by filteringsand. Plankton nets were used to collect medusae fromthe plankton. Samples were fixed both in formalin (formorphological investigations) and in 95% ethanol (formolecular analyses). The species were identified using, amongothers, bibliographic references listed in Table 2.

Where possible, the collected material was also comparedwith type material or other reference specimens obtainedas loans from various museums and private collections(Appendix 1).

A total of 38 nominal species and 6 unidentified specieswere sampled and sequenced. For the latter set, only onecolony or one medusa was found. The available material hasnot allowed description of new species that would meet allthe requirements of the Code of Zoological Nomenclature.However, they still represent the biodiversity of the familyand were therefore kept in the analyses.

Not included in the analysis were sequences from thespecies formerly known as Podocoryna minuta (Mayer 1900b;synonymous with Lizzia blondina Mayer 1900a) and Podoco-rynoides minima (Trinci 1903). Both species belong in theFamily Rathkeidae (Schuchert 2007).

DNA sequencingA 611-bp fragment of the mitochondrial 16S gene was amplifiedusing primers SHA 5"-ACGGAATGAACTCAAATCATGT-3"and SHB 5"-TCGACTGTTTACCAAAAACATA-3"(Cunningham & Buss 1993). Amplification took place underthe following PCR conditions: 1 min at 94 °C, then 35 cyclesof 94 °C for 15 s, 50 °C for 1 : 30 min and 72 °C for 2 : 30 minwith a final extension at 72 °C for 5 min. For some representativesof each of the major clades 231 bp of the nuclear markerElongation Factor1! (EF1!) was also amplified and sequenced.Primers for the EF1! were designed by Lindner et al. (2008).PCR annealing temperature ranged from 55 to 60 °C dependingon the species. Other PCR parameters were as describedabove for the mitochondrial gene.

The PCR product was purified using a QIAquick spin-column purification kit (Qiagen Inc., Valencia, CA). Thepurified PCR product was run on a 2% agarose gel stainedwith ethidium bromide to assay the quantity and quality (i.e.accessory bands) of the product. The purified PCR productwas used as a template for double stranded sequencing.

Phylogenetic analysisThe sequences were first assembled and edited using thesoftware SEQUENCHER 3.0 (Gene Codes Corp., Ann Arbor, MI).They were then aligned using CLUSTALX (Thompson et al.1997). All the alignments were then confirmed and edited byeye in MacClade 4.06 OS X (Maddison & Maddison 2000).A variable region of the mitochondrial 16S gene, comprisinga total of 28 bp was difficult to align in the complete data set,and was removed for some analyses. The nuclear EF1!sequences also contained a highly variable intron that wasdeleted from the analysis.

Phylogenetic analysis of the aligned sequences was performedusing the maximum parsimony optimality criterion in PAUP*version 4.0b10 for Macintosh (Swofford 2001) and themaximum likelihood (ML) optimality criterion in GARLIv0.951.OsX-GUI (Zwickl 2006). Clade stability was assessedby ML bootstrap analyses (Felsenstein 1985) in GARLI (100bootstrap replicates). The ML analyses in GARLI wereperformed using random starting trees and default terminationconditions. The best-fit model of nucleotide substitution foreach gene was a six-parameter model with invariant sites andgamma distribution. The parameters for this model wereestimated by GARLI during each run.

Results and discussionAs described in the Introduction, gene genealogies using themitochondrial 16S gene have been shown to reliably identifyreciprocally monophyletic clades in the Hydrozoa (Schuchert2005; Govindarajan et al. 2005; Miglietta et al. 2007; Miglietta& Lessios 2008; Moura et al. 2008). The 16S gene is less successfulat recovering deeper nodes within the Hydractiniidae (Cun-ningham & Buss 1993). In this article, we will focus mostly onassigning monophyletic groups to nominal species based onthe 16S gene. A complete study of species relationships andthe monophyly of genera require additional data, and will beaddressed in a separate paper (Miglietta et al. manuscript inpreparation). We begin by analysing all of our 16S sequencestogether. Then, we discuss each of four major clades or gradesin turn, bringing to bear nuclear EF1! sequences where available.The nuclear data helped confirm that nuclear and mitochondrialmarkers are identifying genealogically distinct species.

Complete phylogenies of 16S and EF1!!!!The complete 16S data set comprised 233 sequences(Table 1), including 226 members of the Family Hydractiniidaeand 7 outgroup sequences belonging to the Stylasteridae, thesister group of the Hydractiniidae (Cairns 1983a; Cairns &Barnard 1984, 1987; Cartwright et al. in press). After removingthe 28 bp variable region, the aligned sequences included611 bp of the 16S mitochondrial gene. This region included330 constant sites, 194 phylogenetically-informative sites,and 87 autapomorphies.

M. P. M

iglietta et al.•







413

Table 2 Localities where the nominal species of Hydractiniidae herein analized were reported in previous works (and relative references), and localities were the species were collectedduring this work.

Species DistributionLocalities of the species were collected in the present study References

Clava multicornis (Forskal 1775) Mediterranean Sea, West and East Atlantic Ocean

Woods Hole, East USA; Europe Forskal 1775; Broch 1916; Pena Cantero & García Carrascosa 2002; Bouillon et al. 2004

Clavactinia gallensis Thornely 1904 Ceylon; India; Seychelles Gulf of Siam, Thailand Thornely 1904; Annandale 1915; Millard & Bouillon 1973Hydractinia allmanii Bonnevie 1898 Greenland; Arctic New Siberian Island;

Norway; IcelandReykjavík, Iceland; Behring Sea, Aleutian Islands, Alaska

Mayer 1900a; Rees 1956; Naumov 1969; Schuchert 2001

Hydractinia altispina? Hartlaub 1905 False Bay and Lambert’s Bay, South Africa False Bay, South Africa Millard 1955Hydractinia antonii Miglietta 2006 Aleutian Islands, AK, USA Aleutian Islands, Alaska, USA Miglietta 2006Hydractinia calderi? Bouillon, Medel & Peña-Cantero 1997 Mediterranean Sea Santa Caterina, Italy Bouillon, Medel & Pena Cantero 1997; Bouillon et al. 2004Hydractinia laevispina Fraser 1911 California, USA California, USA Fraser 1911Hydractinia echinata (Fleming 1923) Northeastern Atlantic from the Arctic

Seas south to NW AfricaScotland; Denmark; Belgium; Roscoff, France Fleming 1923; Broch 1916; Naumov 1969; Vervoort 1972;

Schuchert 2001, 2008Hydractinia epiconcha Stechow 1907 Kominato, Sagami Bay, Japan Kominato, Nagaki, Japan Stechow 1907; Hirohito 1988Hydractinia fucicola (Sars 1857) Mediterranean Sea to Brittany Torre del Serpe, Otranto, Italy Sars 1857; Allman 1872; Iwasa 1934;

Motz-Kossowska 1905; Bouillon et al. 2004Hydractinia G.M. Gulf of Mexico, Florida, USA Gulf of Mexico, Florida, USA Cunningham & Buss 1993Hydractinia milleri Torrey 1902 From British Columbia to Southern

California, USAVancouver, Canada; Bodega Bay, (CA), Friday Harbor (WA), USA

Torrey 1902; Fraser 1937

Hydractinia multigranosi (Namikawa 1991) Hokkaido (Oshoro Bay), Misaki — Japan Oshoro, Japan Sea coast of Hokkaido, Japan Namikawa 1991Hydractinia n. sp. 1 Monterey Bay (CA), USA Monterey Bay (CA), USA perhaps a new speciesHydractinia n. sp. 2 California, USA California, USA perhaps a new speciesHydractinia n. sp. 3 Alaska, USA Alaska, USA perhaps a new speciesHydractinia n. sp. 4 Ushimato, Seto Inland Sea, Japan Ushimato, Seto Inland Sea, Japan perhaps a new speciesHydractinia polyclina Agassiz 1862 Northwest Atlantic, USA Maine, Woods Hole (MA), USA Agassiz 1862; Buss & Yund 1989; Cunningham et al. 1991Hydractinia pruvoti Motz-Kossowska 1905 Mediterranean Sea Banyuls, France Motz-Kosowska 1905; Bouillon et al. 2004; Iwasa 1934;

Bavestrello et al. 2000Hydractinia rubricata Schuchert 1996 Kaikoura to Dunedin, New Zealand Portobello, Dunedin, New Zealand Schuchert 1996Hydractinia serrata Fraser 1911 West coast of US; Greenland Bering Sea, Alaska; WA, USA Fraser 1911; Naumov 1969; Schuchert 2001Hydractinia sodalis Stimpson 1858 Sagami Bay, Japan Okushiri Is., Hokkaido, Japan Stimpson 1858; Goto 1910; Stechow 1921a; Hirohito 1988Hydractinia symbiolongicarpus Buss & Yund 1989 North West Atlantic, USA Woods Hole (MA), Long Island Sound, USA Buss & Yund 1989; Cunningham et al. 1991Hydractinia symbiopollicaris Buss & Yund 1989 North West Atlantic, USA Woods Hole (MA), USA Buss & Yund 1989; Cunningham et al. 1991Hydractinia uchidai Yamada 1947 Muroran, Hokkaido, Japan Muroran, Pacific coast of Hokkaido, Japan Yamada 1947; Bouillon, Medel & Pena Cantero 1997;

Namikawa, 1994Janaria mirabilis Stechow 1921a Baja California to Panama and Fiji Baja California (CA), USA Stechow 1921a, 1962; Cairns & Barnard 1984Podocoryna n. sp. Kalk Bay, South Africa Kalk Bay, South Africa perhaps a new speciesPodocoryna americana Mayer 1900a Northwest Atlantic Florida; Woods Hole, Long Island, USA Mayer 1900a; Edwards 1972; Mills 1976Podocoryna australis Schuchert 1996 North Island, New Zealand Leigh Marine Reserve, New Zealand Schuchert 1996Podocoryna bella Hand 1961 Portobello, Dunedin, New Zealand Portobello Marine Lab. (in aquarium tank),

Dunedin, New ZealandHand 1961; Kramp 1968; Schuchert 1996

Podocoryna borealis Mayer 1900a Maine, USA; Iceland; British Isles from the Channel coast to Shetland; North Sea; southern and western Norway

Reykjavík, Keflavic, Iceland Mayer 1900a; Edwards 1972; Schuchert 2001



•M



•©




Podocoryna exigua Haeckel 1880 Mediterranean Sea and Britanny Torre del Serpe, Otranto, Italy Haeckel 1880; Bouillon et al. 2004Podocoryna hayamaensis Hirohito 1988 Sagami Bay, Japan Shimoda Bay, Izu Peninsula; Ushimato,

Seto Inland Sea, JapanHirohito 1988

Podocoryna selena Mills 1976 Florida, USA Florida, USA Mills 1976; Cunningham & Buss 1993Stylactaria n. sp. (on Sargassum) Kominato, Boso Peninsula, Japan Kominato, Boso Peninsula, Japan perhaps a new speciesStylactaria carcinicola Hiro 1939 Muroran and Sagami Bay, Japan Ito and Shimoda, Izu Peninsula; Mikimoto

Pearl Island, Toba, Kii Peninsula; Nakagi,Izu Peninsula; Japan

Hiro 1939; Yamada 1959; Hirohito 1988; Namikawa 1997

Stylactaria conchicola Yamada 1947 Muroran, Japan Oshoro, Japan Sea coast of Hokkaido, Japan Yamada 1947; Namikawa et al. 1990; Bouillon et al. 1997Stylactaria hooperii sp. 1 Sigerfoos 1899 Coldspring Harbor, Long Island; Lloyd’s

Harbor, Huntington BayWoods Hole, East USA Sigerfoos 1899; Bouillon, Medel & Peña Cantero 1997;

Pena Cantero & García Carrascosa 2002Stylactaria hooperii sp. 2 Monterey Bay (CA), USA Monterey Bay (CA), USA perhaps a new speciesStylactaria inabai Hirohito 1988 Sagami Bay, Japan Shimoda Bay, Izu Peninsula; Misaki, Japan Hirohito 1988; Namikawa 1991Stylactaria inermis Allman 1871 Mediterranean Sea Torre del Serpe, Otranto, Italy Allman 1871; Iwasa 1934; Bouillon, Medel & Pena

Cantero 1997; Bouillon et al. 2004Stylactaria misakiensis Iwasa 1934 Misaki and Sagami Bay, Japan Shimoda Bay, Izu Peninsula; Sagami Bay, Japan Iwasa 1934; Hirohito 1988; Namikawa 1991Stylactaria reticulata Hirohito 1988 Sagami Bay, Japan Choshi, Boso Peninsula, Japan Hirohito 1988; Bouillon, Medel & Pena Cantero 1997

Species DistributionLocalities of the species were collected in the present study References

Table 2 Continued.T

he complete EF1! data set com

prises 54 sequences including52 m

embers of the fam

ily Hydractiniidae and 2 outgroup

sequences belonging to the Stylasteriidae. After rem

oving anintron of variable length, the aligned sequences included 231 bp.T

his region included 134 constant sites, 86 phylogenetically-inform

ative sites and 11 autapomorphies.

Figure 2A–C

present the maxim

um-likelihood phylogeny

for all 233 sequences. Species assignments for reciprocally

monophyletic groups w

ere made through extensive consulta-

tion with the taxonom

ic literature. Figure 3 shows the E

F1!phylogeny for a subset of sam

ples. This nuclear gene w

as more

difficult to amplify and sequence, but is rem

arkably congruentw

ith the 16S phylogeny. Collection details are presented in

Table 1. Information about each nom

inal species and relevantliterature are presented in Table 2. In several cases, probablespecies w

ere only identified by single sequences that were

diverged from other m

onophyletic groups of taxa, were geo-

graphically disjunct, or were confirm

ed by EF1! (Figs 2A

–4 ).

The P

odocoryna clade Figure 4 show

s a 16S phylogeny of this species group thatincludes the 28 bp variable region excluded in the full phylogenyshow

n in Fig. 2. This phylogeny show

s a strongly supported(100%

bootstrap) group that includes all sequences from species

known to produce fully form

ed medusae —

the definition ofthe genus Podocoryna (see above). T

he genus Podocoryna is para-phyletic, since this strongly supported group also includes onespecies

with

a partially-form

ed, paedom

orphic m

edusa(H

ydractinia pruvoti). These species are discussed in the context of the

phylogeny in Figs 3 and 4.

Podocoryna hayamaensis/H

ydractinia pruvoti.Podocoryna

haya-m

aensis is the only Podocoryna species we found in Japan,

despite the fact that three Podocoryna species were distin-

guished by Em

peror Hirohito (1988) from

the main location

where w

e collected (Sagami Bay, near Shim

oda Bay). AlthoughH

irohito named only one of them

, this is probably neverthelessa case of over-splitting, as H

irohito (1988) often separated newspecies on the basis of their substratum

type. Our discovery

of no other Japanese Podocoryna besides P. hayamaensis is

consistent with the fact that P. hayam

aensis is the only JapanesePodocoryna w

ith a fully-described life cycle (Hirohito 1988),

suggesting that the other two species distinguished by

Hirohito m

ay not be valid. Some of the P. hayam

aensis coloniesfound in the Japanese Inland Sea at U

shimato deviated from

Hirohito’s description of 20 tentacles, since the Inland Sea

colonies had feeding polyps with only six to eight tentacles

(localities in Table 1). How

ever, no genetic differences were

found within this species, w

hich may m

ean these differencesare environm

entally induced.H

ydractinia pruvoti is the only species in the Podocoryna cladew

ithout fully formed m

edusae. The gonophores of this species

M. P. Miglietta et al. • Reconciling genealogical and morphological species in the Hydractiniidae

© 2009 The Authors. Journal compilation © 2009 The Norwegian Academy of Science and Letters • Zoologica Scripta, 38, 4, July 2009, pp403–430 415

Fig. 2 A–C. Maximum likelihood of phylogenetic hypothesis based on partial 16S mitochondrial gene (c. 600 bp) containing all the sequencesused in this study, tree split into three sections A–C. The branch length indicator represents 0.1 substitutions per site. Members of the familyStylasteridae that were used as outgroup are identified in grey. The bracketed names on the right of the phylogeny reflect the taxonomicdecisions made in this article whereas the individual sequences refer to tentative assignments after collection.



develop into medusoids that can be released or not, depend-ing on the circumstances (Schuchert 2008). The eumedu-soids have apparently no functional mouth, but may havetentacle rudiments. Otherwise they are similar to other Podo-coryna medusae and most likely represent a very recent caseof paedomorphosis (Boero & Sara 1987; Cunningham &

Buss 1993). As discussed by Schuchert (2008), H. pruvoti isvery rare, endemic to the Western Mediterranean, and isknown from only a few collections.

Podocoryna carnea/P. exigua. The specific status of P. carnea isespecially important because it is the type species of the

Fig. 2 Continued.



genus, and tends to be the default name given to NorthAtlantic Podocoryna by non-taxonomists, such as ecologistsand developmental biologists (e.g. Masuda-Nakagawa et al.2000; Bumann & Buss 2008). Our results confirm Schuchert’s(2008) judgement that P. carnea and P. exigua are distinct,geographically disjunct species, with P. carnea ranging fromNorway to Denmark, and P. exigua ranging from Brittany in

France to the Mediterranean (Table 1). The type locality ofP. carnea is Norway (Sars 1846), and our single sample collectedfrom the North Sea coast of Denmark (Table 1) matched thecarnea morphotype (see Schuchert 2008). In the 16S phylogeny(Fig. 4), the samples of P. exigua form a tight monophyleticgroup relative to the sample of P. carnea, which is 10 bpdiverged from the P. exigua clade (1.6% p-distance). The

Fig. 2 Continued.



Fig. 3 Maximum likelihood of phylogenetic hypothesis based on the nuclear gene EF1! containing representative of each major clade ofHydractiniidae. Numbers near the nodes indicate the bootstrap value (ML, 100 replicates). Nodes without number indicate that the bootstrapsupport was lower than 50%.



Fig. 4 Maximum likelihood phylogenetic hypothesis based on partial 16S mitochondrial gene of the data set containing sequences of Podocorynaand Clava species, Hydractinia pruvoti, Hydractinia inermis and Hydractinia fucicola. Bootstrap notations are as in Fig. 3.



specific status of P. carnea is also supported by its position asdistinct from P. exigua in the nuclear EF1! phylogeny(Fig. 3).

The importance of properly defining the species status ofthese species is underlined by the fact that two GenBankrecords of P. carnea — one 16S (Collins et al. 2005; GenBankAY512513) and one EF1! sequence (Groeger et al. 1999;GenBank AJ549292) — should both be renamed as P. exiguaaccording to Figs 3 and 4.

Podocoryna americana/P. selena. The 16S sequences of Podocorynafrom New England, New York and Florida all fall into adistinct monophyletic group (Fig. 4), confirming the judgementof Edwards (1972) on the specific status of P. americana. Moststudies on the East Coast of the United States by non-taxonomistshave named their samples P. carnea (e.g. Cunningham & Buss1993; Bridge et al. 2004; Bumann & Buss 2008), and shouldnow refer to P. americana. In contrast, more extensive samplingof P. americana follows Cunningham & Buss (1993) in failingto support the existence of a distinct, P. selena in the Gulf ofMexico, suggesting that P. selena should be subsumed underP. americana. This is in contrast to the situation in Hydractinia,where the Gulf of Mexico samples are strongly diverged(see below).

Podocoryna borealis/P. australis/P. bella/P. sp. We collected P.borealis from Iceland and Scotland. Both the 16S and EF1!phylogenies (Figs 3 and 4) confirm Edwards’s (1972) andSchuchert’s (2008) judgement that the distinct morphologyof the P. borealis medusae sets it apart from the other NorthAtlantic Podocoryna species. The 16S phylogeny showsP. borealis to be a paraphyletic group (Fig. 4), although the EF1!phylogeny in Fig. 3 shows two P. borealis individuals descendedfrom a single common ancestor. Although this may simply bea rooting problem caused by the long branches of H. pruvotiand P. hayamaensis, the paraphyly of P. borealis is confirmed bytwo samples from the Southern hemisphere that are nestedwithin P. borealis in both the 16S and EF1! phylogenies (Figs 3and 4). This includes one sample of P. bella from New Zealandfrom a fish, and one undescribed sample from South Africa(P. sp. Table 1). The remaining Podocoryna samples from NewZealand are P. australis, which form a distinct monophyleticgroup in both the 16S and EF1! phylogenies (Figs 3 and 4).

The observation that P. bella from New Zealand is distinctfrom P. australis collected from Otago Bay confirms Schuchert’s(1996) opinion that P. bella, found growing on the pigfishCongiopodus leucopaecilius is distinct from the more commonP. australis. The 16S sequence of the New Zealand P. bellasample differs only in 4 bp from the Northern P. borealis(0.5%, Fig. 4), thereby falling within the range of intraspecificvariation in P. borealis (Fig. 4). The adult medusa of P. bella isunknown (Schuchert 1996) and the polyps and young

medusae of P. bella are very similar to P. borealis. The similaritiesat the molecular and morphological level of P. borealis andP. bella suggest that also the unknown adult medusa of P. bellawill resemble P. borealis.

An unidentifiable Podocoryna medusa was collected fromSouth Africa (Podocoryna sp. South Africa: Figs 2A and 4).The latter medusa was an immature juvenile, had eighttentacles and was therefore indistinguishable from P. borealisof the same age. The 16S sequence of this sample was 9 bpdiverged from H. borealis (Fig. 4 and 1.4%). Though furthersampling is needed to confirm whether the southern taxaform genealogically-monophyletic groups, the P. borealiscomplex appears to form a closely related, anti-tropical cladein the cold waters of the Northern and Southern Hemispheres.

One prominent Podocoryna species in the North Atlantic,P. areolata collected from Norway, could not be included inthis study as its 16S sequence (GeneBank accession numberAM939651) became available only after all the analyses weredone. A preliminary analysis including P. areolata placed alsothis species safely within the Podocoryna clade (data not shown)(Figs 2A and 4).

The Clava cladeClava multicornis. Clava multicornis colonies generally showthe characteristics of Stylactaria, with periderm-coveredstolons. The medusae are strongly paedomorphic, showingthe sporosac condition of a complete absence of radial canalsand never being released from the colony, so that gametes arereleased directly from sporosacs clustered below the feedingtentacles (Schuchert 2008 and references therein). Unlikemost hydractiniids, including Podocoryna, Clava polyps arenever polymorphic (Schuchert 2008 and references therein).Clava is a monotypic genus whose unusual arrangement ofsporosacs originally placed them in the Family Clavidae (butsee Schuchert 2001). Our sampling of Clava multicornis 16Sconfirms Schuchert’s (2008) judgement that it is found acrossthe North Atlantic, including Iceland (Fig. 4).

Stylactaria inermis/S. fucicola/S. sp. Sargassum. Unlike mostmembers of the Hydractiniidae, Clava multicornis is usuallyfound growing on fucoid algae. In the Mediterranean thereare two Stylactaria species that are also found growing onalgae, namely S. inermis, and S. fucicola. The former is thetype species for the genus Stylactaria. Like Clava, these speciescan also be found on substrata such as rocks, but unlike mosthydractiniids, they are never found on motile substrata suchas gastropod shells occupied by living snails or hermit crabs.These two species do not share Clava’s derived morphology,and have some degree of polymorphism in their polyps.Stylactaria inermis and S. fucicola are very difficult to distinguishmorphologically, a difficulty that is compounded by theirbeing found on the same substrata (Schuchert 2008).



The 16S sequences of S. inermis and S. fucicola show verydeep divergences. The four S. inermis sequences fall into twomonophyletic groups nearly 6% apart from one another (Fig. 4).Each of the S. inermis clades is 5.6% diverged from both theClava clade and our single S. fucicola sequence (Fig. 4), suggestingthat they represent two cryptic species. Finally, a colonygrowing on the algae of the genus Sargassum in Japan (Stylactariasp. 1 on Sargassum, Fig. 4) is 6.3% diverged from Clava,S. inermis, and S. fucicola.

To summarize, all three algae dwelling Stylactaria — whichinclude the type for the genus — plus Clava are between 5.6–6.3% diverged from one another. Although additional nucleardata are needed, these four algae dwelling species probablyform a monophyletic group (Miglietta et al. in preparation).

The deep divergences in these lineages sharply contrastwith those separating the European Podocoryna species(between 1.5% and 2.4% from one another, and 3% divergedfrom the P. Americana) (Fig. 4). More nuclear data andgreater sampling are needed for the algae dwellers. However,for comparison the Podocoryna species, with much smaller16S divergences, are shown by the EF1! phylogeny in Fig. 3to be true genealogical species (see discussion above).

As a final point, it is intriguing that in both the Podocorynaand Clava clades there are divergent, predominantlyMediterranean lineages (see H. pruvoti above and in Figs 3and 4). Even the morphologically homogeneous S. inermis/S.fucicola group has three divergent lineages with distancestwice as great as between most Podocoryna species. The agesof these lineages almost certainly pre-date the Mediterranean,and may be Tethyan remnants (Figs 2A, 3 and 5).

The Japanese misakiensis/inabai/multigranosi clade This Japanese species group is found in shallow waters withpartially developed eumedusoids having few or no tentacles,and periderm-covered stolons. The stolons place them in thepolyphyletic genus Stylactaria. This species group can bedivided into three closely related, geographically disjunctgenealogical species based on congruent 16S and EF1!phylogenies (Figs 3 and 5). These phylogenies show threeclosely related groups, corresponding to geographicalregions and separated by fixed differences. The northerngroup can clearly be assigned to S. multigranosi based on itshost (the gastropod Nassarius multigranosus) and its location onthe northern Japanese island of Hokkaido (Namikawa 1991).

The Central Japanese group is a mix of individualsidentified according to Hirohito’s (1988) diagnosis dependingon their host, with Stylactaria inabai being found on hermitcrabs, and S. misakiensis being found on gastropods. TheEF1! and 16S phylogenies (Figs 3 and 5) show that there areno consistent DNA sequence differences between individualscollected from gastropods and hermit crabs on sandy bottomswith eelgrass at three different locations (Shimoda Marine

Laboratory, Misaki Marine Station, and Nabeta). This stronglysuggests that this group of putative S. inabai and S. misakiensisindividuals should be collapsed into the older S. misakiensis(Iwasa 1934) — shallow-water hydroids found on either gas-tropods or hermit crabs off the Pacific Coast of Central Japan.The genetic distance (p) between S. misakiensis and S. multi-granosi in the mitochondrial gene is 0.5%. To the South, inthe Seto Inland Sea of Japan, a third genealogical species isidentified by the EF1! and 16S phylogenies (Figs 3 and 5;samples 211, 213, 216, 238). These were always small colo-nies found on shallow, mud-dwelling gastropods with no visi-ble reproductive structures. The genetic distances (p)between this third clade and S. misakiensis and S. multigranosi,in the mitochondrial gene, are, respectively, 0.7% and 0.5%

Hirohito (1988) observed consistent morphologicaldifferences between S. misakiensis depending on whether theywere found on gastropod or hermit crab hosts. Thesedifferences appear to be induced by the substratum wherethey are found. For example, the hermit-crab dwellers havetheir periderm-covered stolons in parallel bundles that cangrow past the margin of the shell as the hermit crab growslarger. The gastropod dwellers have reticulate stolons and nevergrow past the margin of the shell. Intra-specific, substratum-induced differences in morphology are common in hydrozoans(see Piraino et al. 1990 for a brief review). The EF1! phylogenyshows the Seto Inland Sea species to be a sister-group tothe Central Japanese S. misakiensis. Since there are nomorphological features on the Inland Sea colonies, they will beconsidered a sub-species of S. misakiensis (Figs 2B, 3 and 6).

The Japanese carcinicola/epiconcha groupA second group of Japanese hydractiniids found off thePacific Coast of Central Japan (variously known as S. carcinicolaand H. epiconcha) can be distinguished morphologicallybecause the formerly discussed S. misakiensis has much smallerpolyps (1–2 mm) than carcinicola/epiconcha (6–10 mm). Thisspecies group illustrates both of the major problems inhydrozoan taxonomy: splitting taxa that are not distinguish-able by genetic data (as was the case with S. misakiensis vs.S. inabai, above); and the inability to morphologicallydistinguish distantly related clades, as described in thefollowing discussion.

More than any other species group, this one shows how thepresence of periderm-free areas on the colony (naked coenosarc)— traditionally used to identify the genus Hydractinia — is amisleading character. Figure 6 shows a 16S phylogeny withH. epiconcha colonies sharing identical haplotypes withS. carcinicola. It should be noted, though, that Hirohito (1988)observed that the apparently naked coenosarc of H. epiconchahad a very thin covering of periderm, consistent with beingplaced in Stylactaria. Although there are two divergentmitochondrial subclades in Fig. 6, a limited number of EF1!



sequences do not support distinct genealogical species(Fig. 3). Individuals from each of the subclades had identicalEF1! sequences (112 H. epiconcha Japan, 089 S carcinicolaJapan) that were 3 bp diverged from a colony bearing thebasal mitochondrial haplotype (105 S carcinicola Japan). Forthis reason all these species will be collapsed under the oldername of H. epiconcha (Stechow 1907), although the genericdesignation will need to be revisited.

More importantly, of a total of 52 colonies morphologicallyidentified as H. epiconcha or S. carcinicola, only 42 are shownin Fig. 6 as H. epiconcha. This is because the remaining 10colonies identified as S. carcinicola (Fig. 2B, samples 189–202,204, 206, 208–210) fall into a distantly related clade that is

close to the American S. hooperi (Sigerfoos 1899; discussed infollowing section). These 10 colonies were morphologicallyindistinguishable from the 22 in Fig. 6 originally identified asS. carcinicola (Fig. 6). This is a very deep divergence, on theorder of millions of years, between colonies that cannot thusfar be distinguished morphologically from one another.

Miscellaneous clades of Hydractinia and StylactariaCarcinicola/hooperi/calderi group (Figs 2B and 3). A morpho-logical resemblance between S. carcinicola and the Atlantic S.hooperi (Sigerfoos 1899) has been noted in the literature(Hirohito 1988). Although most S. carcinicola colonies (22 of32) are now collapsed into H. epiconcha (see discussion above),

Fig. 5 Maximum likelihood phylogenetic hypotheses based on partial 16S mitochondrial gene for the Japanese S. misakiensis andS. multigranosi. Bootstrap notations are as in Fig. 3.



10 S carcinicola colonies fall into a clade that includes aJapanese unidentified species (Hydractinia sp. 4 from the SetoInland Sea at Ushimado), a Mediterranean species (H. calderi),and species on the Western and Eastern Coasts of NorthAmerica. Although Cunningham & Buss (1993) called thespecies found on gastropods in the Monterey Bay California,S. hooperi, this name must be reserved for the populationrepresented by the individual taken from a bivalve in WoodsHole, Massachusetts (205 Stylactaria hooperi USA). In the 16Stree (Fig. 2B), the Woods Hole S. hooperi falls in a clade withthe 10 S carcinicola colonies mentioned above. Although thismight suggest that the Japanese colonies should be named S.

hooperi, limited sampling of EF1! sequences indicates thatthe Japanese S. carcinicola fall outside of a group that includesthe Californian Monterey Bay species, the MassachusettsS. hooperi, and the Italian H. calderi (Fig. 3). Until the NewEngland population can be sampled in greater detail, we willretain the name S. carcinicola for the 10 Japanese colonies.

Hydractinia serrata (Fig. 2B). This species is very common inthe Bering Sea living on gastropods, hermit crabs, and crabcarapaces. One sample has been found as far south as FridayHarbor, Washington, on a hermit crab. There is a second,diverged mitochondrial lineage in the Bering Sea (46 H. serrata

Fig. 6 Maximum likelihood phylogenetichypotheses based on partial 16S mitochondrialgene for the Japanese H. carcinicola. Bootstrapnotations are as in Fig. 3.



USA), that probably represents a cryptic species, but we wereunable to amplify EF1! from H. serrata so confirmation ofa second genealogical species in the Bering Sea will have towait. Like many other hydractiniids that live on hermit crabs,H. serrata grows beyond the lip of the gastropod shell onwhich it is found (See discussion of S. misakiensis/inabaiabove, and H. polyclina below). In H. serrata, though, thisgrowth is taken to an extreme on the hermit crab Labidochirussplendescens. This hermit crab is always found in associationwith H. serrata, and inspection of the shell reveals that almostall of the ‘shell’ is formed from the H. serrata colony, withonly a tiny gastropod shell at its centre (Abrams et al. 1986).Colonies of H. serrata have been collected from Norway(Cunningham, personal observation), but DNA sequencesare not available to confirm the species identity.

Allmanii/antonii/conchicola group (Figs 2B and 3). Also foundin the Bering Sea is Hydractinia allmanii (see Naumov 1969),which is only found on hermit crabs (never on gastropods ortrue crabs). This species does not extend the shell of itshermit crab hosts, and is also found in Iceland (Figs 2B and 3).This species has eumedusoids (Rees 1956; Schuchert 2001)with four radial canals, as does the unusual H. antonii(Miglietta 2006), which is also found in the Bering Sea. H.antonii has a calcified skeleton that is very different from thecalcified skeleton in the distantly related hydractiniid Janaria(see discussion below). In the same clade, though, is theJapanese S. conchicola, which has entirely reduced sporosacreproductive structures, and has opposite host-specificity toH. allmanii, being only found on the living gastropodHomalopoma sagarensis in Hokkaido, Japan (Yamada 1947;Namikawa et al. 1992; Hirohito 1988).

To summarize, this species group is found in the cold-temperate region of the northern hemisphere, including yetanother likely species from the Bering Sea, 128 H. sp. USAin Fig. 2B (H. sp. 1 in Table 2).

Milleri/laevispina/uchidai/altispina group (Figs 2B and 3).Like the allmanii/conchicola group, this clade also has a mix oftaxa with eumedusoids retaining radial canals, and reducedsporosacs with no trace of canals or other medusoid features.This group is found in the northern and southern hemisphere,including two species with eumedusoids (Figs 2B and 3): thealgae-dwelling Japanese S. reticulata and the eumedusoid-bearing South African H. altispina, collected from a livinggastropod. These species are basal to a monophyletic cladewith sporosacs, including the Japanese H. uchidai fromconsolidated sediment in Muroran, Hokkaido, the algae-dwelling individual collected on algae in the Monterey Bay ofCalifornia (078 Hydractinia sp. in Figs 2B and 3).

The sporosac-bearing group includes H. milleri, which iscommon on rocks in the intertidal zone of the Central Californian

Pacific Coast (Bodega Bay). Comparison of 16S and EF1!indicates a second genealogical species collected on a rock indeeper waters off Catalina, California, and off of a floatingdock in Vancouver Canada (Figs 2B and 3). This secondmilleri-like species is most likely H. laevispina (though calledH. californica in Cunningham & Buss 1993). H. laevispina isknown to live on non-motile substrates like rocks and barnacleshells (Fraser 1922). Further sampling of both 16S and EF1!will be required to establish the apparently overlapping rangesof H. milleri and H. laevispina (Figs 2C, 3 and 7).

The echinata species group The species group that has received the most attention as amodel system for laboratory studies of histo-recognition(Lange et al. 1988; Buss & Grosberg 1990; Grosberg et al.1996; Mokady & Buss 1997; Hart & Grosberg 1999;Grosberg 2000; Cadavid et al. 2004, 1997; Müller et al. 2004;Wilson & Grosberg 2004; Nicotra & Buss 2005; Powell et al.2007) and laboratory studies is the complex of species in theNorth Atlantic related to Hydractinia echinata (Fleming 1828).The most important study species have been H. symbiolongicarpusin North America (e.g. Levitan & Grosberg 1993; Grosberget al. 1997; Hart 1997; Buss & Yund 1988), and H. echinata(e.g. Lange et al. 1989; Müller et al. 2004) in Europe. The 16S andEF1! phylogenies agree that the sister group to the H. echi-nata clade is Janaria mirabilis from the Gulf of California. Thisspecies has a highly calcified skeleton, but shares with the echinatagroup the reliance on hermit crabs as hosts (Buss & Yund 1989).

The H. echinata species complex is almost entirely foundliving on gastropod shells occupied by hermit crabs (Buss &Yund 1989). Past efforts at distinguishing this species groupinclude tests of reproductive isolation, morphology andallozymes (Buss & Yund 1989), DNA–DNA hybridization(Cunningham et al. 1991), and 16S DNA (Cunningham &Buss 1993). These studies found significant divergencebetween American and European species, and deep divergencebetween Hydractinia in the Gulf of Mexico and the AtlanticCoast. The current study confirms the divergence of the Gulfof Mexico species, but sampling in Belgium and Denmark hasrevealed a second European lineage that cannot be distinguishedfrom two American species.

Hydractinia symbiolongicarpus group. Buss & Yund (1989)recognized that the Hydractinia found on the hermit crabP. longicarpus in the Long Island Sound was distinct from theHydractinia found on the hermit crab P. pollicaris. It was thusnamed H. symbiolongicarpus. Subsequent DNA–DNAhybridization and 16S phylogenies found that the sister speciesto H. symbiolongicarpus is an undescribed species found in theGulf of Mexico, referred to as H. [GM] in Cunningham et al.(1991) and Cunningham & Buss (1993) and Ferrell (2004). Moreextensive sampling of 16S confirms that H. symbiolongicarpus



is found from Maine on down to North Carolina (Fig. 7,Table 1, samples 41–42, 73–75), and is distinct from severalsamples of H. [GM] from the Gulf of Mexico (43–45).

Echinata/symbiopollicaris/polyclina group. Buss & Yund (1989)named the second species in the Long Island Sound

H. symbiopollicaris after its predominant host P. pollicaris. Theyconsidered H. symbiopollicaris distinct from H. polyclina foundin Maine living on a third hermit crab species — P. acadianus(Agassiz 1862) — based on the reduced mating success of thesetwo forms. Hydractinia symbiopollicaris and H. polyclina werefound by DNA–DNA hybridization and 16S to be significantly

Fig. 7 Maximum likelihood phylogenetic hypothesis based on partial 16S mitochondrial gene of the data set containing sequences ofHydractinia echinata-like species from the North Atlantic. Bootstrap notations are as in Fig. 3.



diverged from H. echinata collected in England and France(Buss & Yund 1989).

The current study has found no genetic differencesbetween H. polyclina and H. symbiopollicaris in either the EF1!or 16S (Figs 3 and 7). Preliminary studies of COI also foundno consistent differences, suggesting that the specific statusof H. symbiopollicaris is in doubt, and may perhaps be sub-sumed under the senior name of H. polyclina L. Agassiz 1862.

While the more extensive sampling in this study confirmsthat H. echinata in France and Scotland are distinct from allAmerican Hydractinia species in both EF1! and 16S (Figs 3and 7), further sampling in Belgium and Denmark (samples010–012, 015, 017, 172, 173) has revealed populations thatare genetically indistinguishable from either H. symbiopollicarisor H. polyclina (Figs 3 and 7).

Until further evidence is found, we conclude that the nameH. polyclina applies to a trans-Atlantic species. This trans-AtlanticH. polyclina has been previously referred to as H. symbiopollicariswhen found on P. pollicaris shells in North America (Cunninghamet al. 1991; Cunningham & Buss 1993) and as H. echinata alongthe North Sea Coast of Belgium and Denmark.

Geographical distributionOf the major clades of Hydractiniidae analysed in this study,11 are found in the Northwest Pacific (Japan), 8 in the NortheastPacific (Canada, California, Alaska, Aleutian Islands), 6 in theMediterranean Sea, 5 in the Northwest Atlantic, 3 in theSouth Pacific (New Zealand), 2 in the South Atlantic (SouthAfrica), one in the Indian Ocean (Thailand). The NorthPacific and especially Japan is the region with the highestdiversity of Hydractiniidae.

The global distribution of clades appears to be affected tosome extent by the ability to disperse through swimmingmedusae. The clade composed of species with swimmingmedusae (genus Podocoryna) has representatives around theworld, from Japan to the Antipodes, Arctic and Atlantic. Itappears to have achieved this broad distribution relativelyrapidly, with a fairly recent ancestor compared to most othermajor clades in Fig. 2. For example, the most recent ancestorof its sister clade is twice as old (the Clava/S. fucicola/S. inermisgroup). Even further evidence of the dispersal ability of freeswimming medusae is a very recent Podocoryna species groupis found in Iceland, New Zealand, and South Africa (P. borealis/P. bella/P. sp. 1 SA).

On the other hand, H. polyclina is found on both sides of theNorth Atlantic despite reproducing with non-swimmingsporosacs and being found living on hermit crabs. This ismost likely a case of rafting by the host hermit crabs on algalrafts (Wares & Cunningham 2001). Algal rafting also explainsthe trans-Atlantic range of Clava multicornis, which also hasnon-swimming sporosacs, but is usually found living directlyon seaweed. Other species groups lacking swimming larvae

are broadly distributed geographically even in the absence ofswimming medusae (e.g. the H. allmanii/S. conchicola group, theS. hooperi (S. carcinicola group)). In these cases, the main expla-nation for their broad range appears to be their relatively old age.

Concluding remarksThis study shows the efficacy of using a combination ofmitochondrial and nuclear markers to begin to untangle speciescomplexes in the morphologically plastic Hydrozoa. Oneimmediate contribution is to identify the correct names to beused for the species most often used as model organisms forecological, physiological and developmental studies. Moststudies of Atlantic Podocoryna have referred to P. carnea. In America,most of these studies have been carried out on material thatshould be referred to as P. americana (e.g. Cunningham &Buss 1993; Bridge et al. 2004; Baumann & Buss 2008;Masuda-Nakagawa et al. 2000), and in Europe, the studies bySchmid’s group have been carried out on P. exigua. Because ofthe work of Buss & Yund (1989), material in American studieshave been correctly identified as H. symbiolongicarpus orH. polyclina. In Europe, where Hydractinia is most widely usedas a model organism, the situation is very complicated. An essayon H. echinata as a model organism (Frank et al. 2001)mentions that H. echinata can be obtained from laboratoriesin Plymouth, England, Roscoff France, and HelgolandGermany. Our study shows that these represent two differentspecies: H. echinata (Plymouth and Roscoff) and H. polyclina(Helgoland, Germany).

Few of the taxa we defined were not monophyletic, but hadfixed differences between them (e.g. Stylactaria multigranosi andS. misakiensis). In another case the mitochondrial and nuclear genesdid not agree in the identifications of taxa (i.e. S. hooperi fromEast and West California). Besides these few exceptions, the cri-terion of monophyly we adopted worked well in defining taxa.

Finally, this work sets the stage for a generic revision of theHydractiniidae. The type for the Family is Hydractinia echinata(Fleming 1828). While much of the phylogeny is unresolved,it appears that the closest relatives to the H. echinata clade aretwo morphologically very distinctive genera: Hydrissa andJanaria (the latter having a fully-calcified skeleton. Thismeans that the proposal by Bouillon et al. (1997), Boero et al.(1998) and Bouillon et al. (2006) to collapse Hydractinia, Stylactariaand Podocoryna into the single genus of ‘Hydractinia’ is notpractical without eliminating clearly distinct genera such asJanaria. This means that several new monophyletic generawill have to be named on the basis of more intensive phylogeneticanalysis and more sequence data.

AcknowledgementsThis work would have not been possible without the tremendoushelp of many people who assisted in the field and/or sentspecimens. We are especially grateful to, H. Namikawa,



S. Piraino, F. Boero, A. Govindarajan, A. Lindner, D. Calder,A. Brinkmann-Voss, L.A. Henry and S. Cairns for providingsamples and/or helping in the field. C. Gravili helped withbibliographic aspects of the article. MPM is grateful to Y.and Y. Hirano for their tremendous support in Japan, toL. McNelly for help in the laboratory, and to A. Faucci.S. Piraino, D. Calder, A. Lindner, F. Boero, H. Namikawafor discussion and suggestions at different stages of thiswork.

This work was supported by the National Science FoundationPEET Grant No. DEB-9978131 A000 to CWC.

ReferencesAbrams, P., Nyblade, C. & Sheldon, S. (1986). Resource partitioning

and competition for shells in a subtidal hermit crab species assemblage.Oecologia, 69, 429–445.

Agassiz, A. (1862). The Acalephan fauna of the southern coast ofMassachusetts (Buzzard’s Bay). Proceedings of the Boston Society ofNatural History, 8, 224–226.

Allman, G. J. (1871–1872). A monograph of the Gymnoblastic or TubularianHydroids, I–II. London: Ray Society.

Annandale, N. (1915). Fauna of Chilka Lake. The Coelenterata ofthe lake, with an account of the Actiniaria of brackish water in theGangetic delta. Memoirs of the Indian Museum, 5, 65–114.

Baum, D. A. & Shaw, K. L. (1995). Genealogical perspectives on thespecies problem. Experimental and Molecular Approaches to PlantBiosystematics, 53, 289–303.

Bavestrello, G., Puce, S., Cerrano, C. & Balduzzi, A. (2000). Lifehistory of Perarella schneideri (Hydrozoa, Cytaeididae) in theLigurian Sea. Scientia Marina, 64, 141–146.

Blackstone, N. W. (1996). Gastrovascular flow and colony developmentin two colonial hydroids. Biological Bullettin, 190, 56–68.

Blackstone, N. W. & Buss, L. (1993). Experimental heterochrony inhydractiniid hydroids: why mechanisms matter. Journal ofEvolutionary Biology, 6, 307–327.

Boero, F. & Sara, M. (1987). Motile sexual stages and evolution ofLeptomedusae (Cnidaria). Bollettino Di Zoologia, 54, 131–139.

Boero, F., Bouillon, J. & Piraino, S. (1998). Heterochrony, genericdistinction and phylogeny in the family Hydractiniidae. ZoologischeVerhandelingen. Leinden, 323, 25–36.

Bouillon, J. (1980). Hydromedusae from the waters surroundingLaing Islands, Papua New Guinea. 3. Anthomedusae. Filifera(Hydrozoa, Cnidaria) [Hydroméduses de la mer de Bismarck(Papouasie Nouvelle-Guinée) partie 3: Anthomédusae-Filifera(Hydrozoa-Cnidaria)]. Cahiers de Biologie Marine 21 (3), 307–344.

Bouillon, J., Medel, D. & Pena Cantero, A. L. (1997). The taxonomicstatus of the genus Stylactaria Stechow, 1921 (Hydroidomedusae,Anthomedusae, Hydractiniidae), with the description of a newspecies. Scientia Marina, 61, 471–486.

Bouillon, J., Medel, M. D., Pagès, F., Gili, J. M., Boero, B. & Gravili,C. (2004). Fauna of the Mediterranean Hydrozoa. Scientia Marina,68 (Suppl. 2), 1–448.

Bouillon, J., Gravili, C., Pagès, F., Gili, J.-M. & Boero, F. (2006). Anintroduction to Hydrozoa. Mémoirs Du Muséum National d’HistoireNaturelle, 194, 1–591.

Bridge, D., Ha, C., Nemir, A., Renden, A., Rorick, M., Shaffer, A.,Underkoffler, D., Willis, A. & Martinez, D. (2004). Variations on

a theme? Polyp and medusa development in Podocoryna carnea.Hydrobiologia, 530 (1), 299–307.

Broch, H. (1916). Hydroida. (Part I). Danish Ingolf Expedition, 5, 1–66.Bumann, D. & Buss, L. (2008). Nutritional physiology and colony

form in Podocoryna carnea (Cnidaria: Hydrozoa). InvertebrateBiology, doi:10.1111/j.1744-7410.2008.00135.X

Buss, L. W. & Grosberg, R. K. (1990). Morphogenetic basis forphenotypic differences in hydroid competitive behavior. Nature,343, 63–66.

Buss, L. W. & Yund, P. O. (1988). A Comparison of recent andhistorical populations of the colonial hydroid Hydractinia. Ecology,69, 646–654.

Buss, L. W. & Yund, P. O. (1989). A sibling species group of Hydractiniain the north-eastern United-States. Journal of the Marine BiologicalAssociation of the United Kingdom, 69, 857–874.

Cadavid, L. F., Powell, A. E., Nicotra, M. L., Moreno, M. & Buss, L. W.(2004). An invertebrate histocompatibility complex. Genetics, 167,357–365.

Cairns, S. D. (1983a). A generic revision of the Stylasterinae(Coelenterata: Hydrozoa). Part 1. Description of the genera.Bulletin of Marine Science, 33, 427–508.

Cairns, S. D. (1987). Evolutionary trends in the Stylasteridae(Cnidaria, Hydrozoa), pp. 257–274. In J. Bouillon, F. Boero, F.Cicogna & P. F. S. Cornelius (Eds) Modern Trends in the Systematics,Ecology, and Evolution of Hydroids and Hydromedusae. Oxford:Clarendon Press.

Cairns, S. D. & Barnard, J. L. (1984). Redescription of Janariamirabilis, a calcified hydroid from the eastern Pacific. BulletinSouthern California Academy of Sciences, 83, 1–11.

Calder, D. R. (1988). Shallow water hydroids of the Bermuda: theAthecates. Contribution 148. Toronto: Royal Ontario Museum ofLive Sciences.

Cartwright, P., Bowsher, J. & Buss, L. W. (1999). Expression of aHox gene, Cnox-2, and the division of labor in a colonial hydroid.Proceeding of the Natural Academy of Science USA, 96, 2183–2186.

Cartwright, P., Evans, N., Dunn, C. W., Marques, A. C., Miglietta,M. P., Schuchert, P. & Collins, A. G. (2008) Phylogenetics ofHydroidolina (Hydrozoa, Cnidaria). Journal of Marine BiologicalAssociation of the United Kingdom. doi:10.1017/S0025315408002257

Collins, A. G., Winkelmann, S., Hadrys, H. & Schierwater, B. (2005).Phylogeny of Capitata and Corynidae (Cnidaria, Hydrozoa) in lightof mitochondrial 16S rDNA data. Zoologica Scripta, 34 (1), 91–99.

Cunningham, C. W. & Buss, L. W. (1993). Molecular evidence formultiple episodes of paedomorphosis in the family Hydractiniidae.Biochemical Systematics and Ecology, 21, 57–69.

Cunningham, C. W., Buss, L. W. & Anderson, C. (1991). Molecularand geologic evidence of shared history between hermit crabs andthe symbiotic genus Hydractinia. Evolution, 45, 1301–1316.

Edwards, C. (1972). The hydroids and the medusae Podocorynaareolata, P. borealis and P. carnea. Journal of the Marine BiologicalAssociation of the United Kingdom, 52, 97–144.

Felsenstein, J. (1985). Confidence limits on phylogenies: anapproach using the bootstrap. Evolution, 39 (4), 783–791.

Ferrell, D. L. (2004). Fitness consequences of allorecognition-mediated agonistic interactions in the colonial hydroid Hydractinia[GM]. Biological Bulletin, 206, 173–187.

Fleming, J. (1828). A history of British animals, exhibiting thedescriptive characters and systematical arrangement of the generaand species of quadrupeds, birds, reptiles, fishes, Mollusca and



Radiata of the United Kingdom. Ball and Bradfute, Edimburg,Edinburgh, pp. 1–565.

Fleming, J. (1923). Gleanings of natural history, gathered on thecoast of Scotland during a voyage in 1821. Edinburgh PhilosophicalJournal, 8, 294–303.

Forskal, P. (1775). Descriptiones animalium quae in itinere orientaliobeservavit, post mortem editit Carstern Niebuhr. Ex officinaMölleri Haunie, 20+XXIV+15.

Frank, U., Leitz, T. & Muller, W. A. (2001). The hydroid Hydractinia: aversatile, informative cnidarian representative. Bioessays, 23, 963–971.

Fraser, C. M. (1911). The hydroids of the west coast of NorthAmerica. With special reference to those of the Vancouver Islandregion. Bulletin from the Laboratories of Natural History of the StateUniversity of Iowa, 6, 39–48, pls 1–8.

Fraser, C. M. (1922). A new Hydractinia and other west coasthydroids. Contributions to Canadian Biology, 1, 97–100, pls 1–2.

Fraser, C. M. (1937). Hydroids of the Pacific Coast of Canada and theUnited States (pp. 208). Toronto: The University of Toronto Press.pls 1–44.

Goto, S. (1910). On two species of Hydractinia living in symbiosis witha hermit crab. The Journal of Experimental Zoology, 9, 469–496.

Govindarajan, A. F., Halanych, K. M. & Cunningham, C. W. (2005).Mitochondrial Evolution and Phylogeography in the HydrozoanObelia geniculata. Marine Biology, 146, 213–222.

Groeger, H., Callaerts, P., Gehring, W. J. & Schmid, V. (1999). Geneduplication and recruitment of a specific tropomyosin into striatedmuscle cells in the jellyfish Podocoryne carnea. The Journal of Experi-mental Zoology, 285, 378–386.

Grosberg, R. K. (2000). Mate selection and the evolution of highlypolymorphic self/nonself recognition genes. Science, 289, 2111–2114.

Grosberg, R. K., Levitan, D. R. & Cameron, B. B. (1996). Evolutionarygenetics of allorecognition in the colonial hydroid Hydractiniasymbiolongicarpus. Evolution, 50, 2221–2240.

Grosberg, R. K., Hart, M. W. & Levitan, D. R. (1997). Is allorecognitionspecificity in Hydractinia symbiolongicarpus controlled by a single gene?Genetics, 145, 857–860.

Haeckel, E. (1880). System der Acraspeden. 2te Hälfte des Systemsder Medusen. Mit: Anhang zum System der Medusen. AchtNachträge zur Vervollständigung des Systems. Denksch MedicineNature Gesellschaft Jena, 2 (2), 361–672.

Hand, C. (1961). A new species of athecate hydroid, Podocoryna bella(Hydractiniidae) living on the pig-fish. Congiopodus leucopaecilus.Transactions of the Royal Society of New Zealand, 1 (5), 91–94.

Hart, M. W. (1997). Population structure and colony fusion amonghydroids. American Zoologist, 37, 130A.

Hart, M. W. & Grosberg, R. K. (1999). Kin interactions in a colonialhydrozoan (Hydractinia symbiolongicarpus): population structure ona mobile landscape. Evolution, 53, 793–805.

Hiro, F. (1939). Notes on the animals found on Macrocheira kaempferide Haan. III. Hydroids. Annotnes Zoology Japanese, 18, 167–176.

Hirohito Emperor of Japan (1988). The hydroids of Sagami Baycollected by his Majesty the emperor of Japan. Biological Laboratoryof the Imperial Household (pp. 179). Tokyo, pls 1–4.

Iwasa, M. (1934). Revision of Stylactis and its allied genera, withdescription of Stylactella (Stylactis) yerii n.sp. Journal of FacultadSciences Hokkaido Imp University, 2, 241–277.

Kramp, P. L. (1922). Kinetocodium danae n.g., n.sp. a new gymnoblastichydroid parasitic in pteropod. Meddr Dansk Naturhist ForenKøbenhavn, 74, 1–21.

Kramp, P. L. (1968). The Hydromedusae of the Pacific and IndianOceans. Sections II and III. Dana Report, 72, 1–200.

Lange, R., Plickert, G. & Müller, W. A. (1988). Histoincompatibilityin a low invertebrate, Hydractinia echinata: analysis of the mechanismof rejection. Journal of Experimental Zoology, 249 (3), 284–292.

Levitan, D. R. & Grosberg, R. K. (1993). The analysis of paternityand maternity in the marine hydrozoan Hydractinia symbiolongicarpususing randomly amplified polymorphic DNA (rapd) markers.Molecular Ecology, 2, 315–326.

Linder, A., Cairns, S. D. & Cunningham, C. W. (2008). From offshoreto onshore: multiple origins of shallow-water corals from deep-seaancestors. Plos ONE, 3 (6), e2429. doi: 10.1371/journal.pone.0002429.

Maddison, D. R. & Maddison, W. P. (2000). MacClade Version 4:Analysis of Phylogeny and Character Evolution. Sunderland Massa-chusetts: Sinauer Associates.

Masuda-Nakagawa, L. M., Gröger, H., Aerne, B. L. & Schmid, V.(2000). The HOX-like gene Cnox2-Pc is expressed at the anteriorregion in all life cycle stages of the jellyfish Podocoryne carnea.Development Genes and Evolution, 210 (3), 151–156.

Mayer, A. G. (1900a). Descriptions of new and little-known medusaefrom the western Atlantic. Bulletin of the Museum of ComparativeZoology of Harvard, 37, 1–9, pls 1–6.

Mayer, A. G. (1900b). Some medusae from the Tortugas, Florida.Bulletin of the Museum of Comparative Zoology of Harvard, 37, 13–82, pls 1–44.

Miglietta, M. P. (2006). Hydractinia antonii sp. nov: a new, partiallycalcified hydractiniid (Cnidaria, Hydrozoa, Hydractiniidae) fromAlaska. Journal of Marine Biological Association of the United Kingdom,86, 993–996.

Miglietta, M. P. & Lessios, A. H. (2008). A silent invasion. BiologicalInvasions. http://Dx Doi Org/10 1007/S10530–, 008–9296–0.

Miglietta, M. P., Piraino, S., Kubota, S. & Schuchert, P. (2007).Species in the genus Turritopsis (Cnidaria, Hydrozoa): a molecularevaluation. Journal of Zoological Systematics and EvolutionaryResearch, 45, 11–19.

Millard, N. A. H. (1955). New species of Hydrozoa from SouthAfrica. Annals of the South African Museum, 41, 215–222.

Millard, N. A. H. (1975). Monograph on the Hydroida of southernAfrica. Annals of the South African Museum, 68, 1–513. Mills,1976.

Millard, N. A. H. & Bouillon, J. (1973). Hydroids from the Seychelles(Coelenterata). Annales Du Musée Royal de l’Afrique Centrale, SérieIn-8°, Sciences Zoologiques, 206, 1–106, pls 1–5.

Mills, C. E. (1976). The association of hydractiniid hydroids andhermit crabs, with new observations from North Florida. In G. O.Mackie (Ed.) Coelenterate Ecology and Behavior (pp. 467–476). NewYork: Plenum Press.

Mokady, O. & Buss, L. W. (1997). Genetics of allorecognition inHydractinia. Genetics, 145, 861–861.

Motz-Kossowska, S. (1905). Contribution à la connaissance deshydraires de la Méditerranée occidentale. I. Hydraires gymno-blastiques. Archives de Zoologie Expérimentale et Générale, 4me Série,3, 39–98.

Moura, C. J., Harris, D. J., Cunha, M. R. & Rogers, A. D. (2008).DNA barcoding reveals cryptic diversity in marine hydroids(Cnidaria, Hydrozoa) from coastal and deep-sea environments.Zoologica Scripta, 37, 93–108.

Müller, W. A., Regina, T. & Uri, F. (2004). Totipotent migratorystem cells in a hydroid. Developmental Biology, 275 (1), 215–224.



Namikawa, H. (1991). A new species of the genus Stylactaria (Cnidaria,Hydrozoa) from Hokkaido, Japan. Zoological Science, 8, 805–812.

Namikawa, H. (1994). Four species of hydractiniid hydroids from offKushiro, Hokkaido, Japan. Memoirs of the National Science Museum,27, 79–86.

Namikawa, H. (1997). Stylactaria carcinicola (Hiro, 1939) (Hydrozoa:Hydractiniidae) from Suruga Bay, Japan. National Science MuseumMonographs, 12, 11–18.

Namikawa, H., Kubota, S. & Mawatari, S. F. (1990). Redescriptionof Stylactaria uchidai (Yamada, 1947), comb. nov. (Hydrozoa:Hydractiniidae) in Hokkaido, Japan. Proceedings of the JapaneseSociety for Systematic Zoology, 42, 2–9.

Namikawa, H., Kubota, S. & Mawatari, S. F. (1992). Redescriptionof Stylactaria conchicola (Yamada, 1947), comb. nov. (Hydrozoa:Hydractiniidae) from Hokkaido, Japan. Hydrobiologia, 231, 69–76.

Naumov, D. V. (1969). Hydroids and Hydromedusae of the USSR. IsraelProgram for scientific translation (pp. 463). Jerusalem.

Nicotra, M. L. & Buss, L. W. (2005). A test for larval kin aggregations.Biological Bulletin, 208, 157–158.

Pena Cantero, A. L. & Garcia Carrascosa, A. M. (2002). The benthichydroid fauna of the Chafarinas Islands (Alboran Sea, westernMediterranean). Zoologische Verhandelingen, 337, 1–180.

Picard, J. (1958). Tregoubovia n. gen. atentaculata n.sp. NouvelleAnthoméduse, dépourvue de tentacules récolté dans le planctonprofond de Villefranche-sur-mer. Rapports et Proces-Verbaux DesRéunions Commision Internationale Pour l’Exploration Scientifique dela Mer Méditerranee, Monaco, 14, 185–186.

Piraino, S., Morri, C. & Boero, F. (1990). Plasticità intraspecifica nelleidromeduse (Cnidaria: Anthomedusae, Leptomedusae): risposte dellafase polipoide a diverse condizioni ambientali. Oebalia, 16 (1), 383–394.

Powell, A. E., Nicotra, M. L., Moreno, M. A., Lakkis, F. G.,Dellaporta, S. L. & Buss, L. W. (2007). Differential effect ofallorecognition loci on phenotype in Hydractinia symbiolongicarpus(Cnidaria: Hydrozoa). Genetics, 177, 2101–2107.

Rees, W. J. (1956). On three northern species of Hydractinia. Bulletinof the British Museum (Natural History), 3, 351–362.

Sars, M. (1846). Fauna littoralis Norvegiae, 1. Heft: Ueber dieFortpflanzungsweise der Polypen. Johann Dahl, Christiania, 1–94.plates.

Sars, M. (1857). Bidrag til kundskaben om middelhavets Littoral-Fauna,Reisebemaerkninger fra Italien. I. Classis: Polypi. Nyt Magazin forNaturvidenskaberne, 9, 110–164.

Schierwater, B., Murtha, M., Dick, M., Ruddle, F. H. & Buss, L. W.(1991) Homeoboxes in cnidarians. Journal of Experimental Zoology,260, 413–416.

Schuchert, P. (1996). The marine fauna of New Zealand: athecatehydroids and their medusae (Cnidaria: Hydrozoa). New ZealandOceanographic Institute Memoir, 106, 1–159.

Schuchert, P. (2001). Hydroids of Greenland and Iceland (Cnidaria,Hydrozoa). Meddelelser Om Grønland, Bioscience, 53, 1–184.

Schuchert, P. (2005). Species boundaries in the hydrozoan genusCoryne. Molecular Phylogenetics and Evolution, 36, 194–199.

Schuchert, P. (2007). The European athecate hydroids and theirmedusae (Hydrozoa, Cnidaria): Filifera Part 2. Revue Suisse deZoologie, 114, 195–396.

Schuchert, P. (2008). The European athecate hydroids and theirmedusae (Hydrozoa, Cnidaria): Filifera Part 3. Revue Suisse deZoologie, 115, 221–302.

Sigerfoos, C. P. (1899). A new hydroid from Long Island Sound.American Naturalist, 33, 801–807.

Stechow, E. (1907). Neue japanische Athecata und Plumulariidaeaus der Sammlung Dr. Doflein. Zoologischer Anzeiger, 32, 192–200.

Stechow, E. (1921a). Neue Gruppen skelettbildender Hydrozoenund Verwandtschaftsbeziehungen rezenter und fossiler Formen.Verhandlungen der Deutschen Zoologischen Gesellschaft, 26, 29–31.

Stechow, E. (1921b). Neue Genera und Species von Hydrozoen undanderen Evertebraten. Archiv für Naturgeschichte, 87, 248–265.

Stechow, E. (1962). Ueber skelettbildende Hydrozoen. ZoologischerAnzeiger, 169, 416–428. Stimpson, 1858.

Stimpson, W. (1858). Prodromus descriptionis animalium evertebra-torum, quae in Expeditione ad Oceanum Pacificum Septentrionalem,a Republica Federata missa, Cadwaladaro Ringgoid et JohanneRodgers Ducibus, observavit et descripsit W. Stimpson. P. VII,Crustacea anomoura. Proceedings of the Academy of Natural Sciencesof Philadelphia, 1858, 225–252.

Swofford, D. L. (2001). PAUP* Phylogenetic Analysis Using Parsimony(*and Other Methods). Version 4. Sunderland, Massachusetts:Sinauer Associates.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &Higgins, D. G. (1997). The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by qualityanalysis tools. Nucleic Acids Research, 25, 4876–4882.

Thornely, L. A. (1904). Report on the Hydroida collected by professorHerdman, at Ceylon, in 1902. Report to the Government of Ceylon on thepearl oyster fisheries of the Gulf of Manaar, 2 (no. Suppl. Rep. 8), 107–126.

Torrey, H. B. (1902). The Hydroida of the Pacific Coast of NorthAmerica. University of California Publications. Zoology, 1, 1–104, pls 1–11.

Trinci, G. (1903). Di una nuova specie di Cytaeis gemmante del Golfodi Napoli. Mitteilungen aus der Zoologischen Station zu Neapel, 16, 1–34.

Van Beneden, P. J. (1867). Recherches sur la faune littorale deBelgique: Polypes [Research on the Belgian littoral fauna: polyps].Mém De L’acad Roy De Belg, 36, 1–226.

Vervoort, W. (1972). Hydroids from the Theta, Vema and Yelchocruises of the Lamont-Doherty geological observatory. ZoologischeVerhandelingen, 120, 1–247.

Wares, J. P. & Cunningham, C. W. (2001). Phylogeography andhistorical ecology of the North Atlantic intertidal. Evolution, 55,2455–2469.

Wilson, A. C. C. & Grosberg, R. K. (2004). Ontogenetic shifts infusion-rejection thresholds in a colonial marine hydrozoan,Hydractinia symbiolongicarpus. Behavioral Ecology and Sociobiology,57, 40–49.

Yamada, M. (1947). On two new species of athecate hydroid Stylactisfrom Hokkaido. Journal of the Faculty of Science. Hokkaido University,9, 383–387.

Yamada, M. (1959). Hydroids from the Japanese Inland Sea, Mostlyfrom Matsuyama and its vicinity. Journal of the Faculty of Science,Hokkaido University Series 6, Zoology, 14 (1), 51–63.

Yund, P. O., Cunningham, C. W. & Buss, L. W. (1987). Recruitment andpostrecruitment interactions in a colonial hydroid. Ecology, 68, 971–982.

Zwickl, D. J. (2006). Genetic algorithm approaches for the phylogeneticanalysis of large biological sequence datasets under the maximum like-lihood criterion. Available via www.bio.utexas.edu/faculty/antisense/garli/Garli.html. PhD Dissertation. The University of Texas at Austin.



Appendix 1 Besides the newly collected specimens (see Table 1), the following specimens from museum or private collections were examinedand used to compare and identify our material.

Species specimen accession code Museum

Hydrissa sodalis colony male, 29 May 1952 Tschuba Museum — JapanHydractinia cryptogonia prep. 60 70 Tschuba Museum — JapanHydractinia granulata prep. 7496. Sp # 1682 — Paratype Tschuba Museum — JapanHydractinia granulata sp # 1685 — Holotype Tschuba Museum — JapanPodocorella minoi Hydr. 2569. Prep 4877-4880. Hyrd. # 3795 Tschuba Museum — JapanPodocoryna hayamamensis sp# 2573. Prep. 1345-13456. Hydr. # 2620. Prep # 8094-8110

— Paratype. Hydr. # 2600. Various slidesTschuba Museum — Japan

Stylactaria halecii Holotype and Paratype Tschuba Museum — JapanStylactaria misakiensis Holotype and Paratype Tschuba Museum — JapanStylactaria spiralis Hydr. 3757. Various prepared slides Tschuba Museum — JapanStylactaria yerii Hydr. # 3787 Tschuba Museum — JapanStylactari brachiurae Holotyype. Prep. 88.99. Hydr. 3022# 8687 — Paratype. Various

prepared slidesTschuba Museum — Japan

Stylactaria inabai Hydr. 3888 — Paratype, Hydr 3888 (eumeduoids), Hydr. 3888 prep 10499-10495. Hydr. 1748. Paratype. Hydr 1759

Tschuba Museum — Japan

Stylactaria monoon Hydr. 1723, Paratype. Hydr 1722, Paratype Tschuba Museum — JapanStylactaria reticulata Prep. No 21211, 21 22. Hydr. 30017, Paratype Tschuba Museum — JapanStylactaria spinipapillaris Hydr. 1725, 1730, 1724 Tschuba Museum — JapanHydrocorella africana H 408, H 481, H 2416, H 2751, 2433, 2442 Cape Town Natural History Museum — SAHydractinia altispina H 407, H 1823, H 1853, H 2651, H 2553, H 3503,

H 87, H 88, H 97, H 123, 776R, CP 258, SAM 114BCape Town Natural History Museum — SA

Hydractinia caffaria H. 3504, H 2928 Cape Town Natural History Museum — SAClavactinia multitentaculata H 2695, H 389, TMI 61A, TME 61 Cape Town Natural History Museum — SAHydractinia marsupalia H 3503, H 2416, FAL209D Cape Town Natural History Museum — SAHydractinia sp. H 1995 Cape Town Natural History Museum — SAHydractinia sp. H 2356, H 2358, H 2883, H 2538, H 2357 Cape Town Natural History Museum — SAClavactinia gallsensis H 503 Cape Town Natural History Museum — SAClavactinia multitentaculata H 1867, H 389 Cape Town Natural History Museum — SAHydractinia kaffaria H 2654, H 92 Cape Town Natural History Museum — SACyteis nassa H 1739, H 122 Cape Town Natural History Museum — SAHydractinia canalifera H97 CP332 Cape Town Natural History Museum — SAPodocoryna carnea Si15 Cape Town Natural History Museum — SAHydractinia polyclina USNM 25503 Smithsonian Institute — DC — USAHydractinia milleri USNM 43525 Smithsonian Institute — DC — USAHydractinia aggragata USNM 71134 Smithsonian Institute — DC — USAHydractinia sodalis USNM 76888 Smithsonian Institute — DC — USAHydractinia allmani USNM 70980 Smithsonian Institute — DC — USAHydractinia bayeri USNM 100198 Smithsonian Institute — DC — USAHydractinia echinata USNM 42534 Smithsonian Institute — DC — USAHydractinia echinata USNM 43897 Smithsonian Institute — DC — USAHydractinia sp. USNM 89155 Smithsonian Institute — DC — USAHydractinia sp. USNM 68775 Smithsonian Institute — DC — USAHydractinia bedofii ? USNM 42673 Smithsonian Institute — DC — USAHydractinia monocarpa USNM 16535 Smithsonian Institute — DC — USAHydractinia serrata C-8 Yale Peabody Museum — CT — USAHydractinia allmani C-8 Yale Peabody Museum — CT — USAHydractinia allmani/serrata L-28 Yale Peabody Museum — CT — USAHydractinia allmani L-28 Yale Peabody Museum — CT — USAHydractinia serrata L-29 Yale Peabody Museum — CT — USAHydractinia serrata Q-31 Yale Peabody Museum — CT — USAHydractiniidae Q-31 Yale Peabody Museum — CT — USAHydractinia serrata male/female Q-31 Yale Peabody Museum — CT — USAHydractinia serrata female S-32 Yale Peabody Museum — CT — USAHydractinia serrata S-32 Yale Peabody Museum — CT — USAHydractinia allmani U-30 Yale Peabody Museum — CT — USAHydractinia californica Santa Catalina, California, USA Yale Peabody Museum — CT — USAHydractinia milleri Bodega Bay, California, USA Yale Peabody Museum — CT — USAHydractinia serrata Coll: J. Marks Yale Peabody Museum — CT — USAHydractinia serrata Bering sea Yale Peabody Museum — CT — USAHydractinia uchidai Hokkaido. Coll Shin Kubota Yale Peabody Museum — CT — USAComplete Hydractiniidae collection Various Speciemens and Localities Royal Ontario Museum — CanadaComplete private collection Various Speciemens and Localities Boero Laboratory — Italy

Reconciling genealogical and morphological species in a worldwide study of the Family Hydractiniidae...

Documents

Transcript of Reconciling genealogical and morphological species in a worldwide study of the Family Hydractiniidae...