Parallel evolution of larval morphology and habitat in thesnail-killing fly genus Tetanocera

E. G. CHAPMAN,* B. A. FOOTE,* J. MALUKIEWICZ� & W. R. HOEH*

*Evolutionary, Population, and Systematic Biology Group, Department of Biological Sciences, Kent State University, Kent, OH, USA

�Program in Biological Anthropology, Department of Anthropology, Kent State University, Kent, OH, USA

Introduction

Detecting significant correlations among ecological and

morphological traits is one of the primary objectives of

comparative biology (Harvey & Pagel, 1991; Martins &

Hansen, 1996; Armbruster, 2002; Felsenstein,

2004). Phylogenetic comparative methods (e.g.

Felsenstein, 1985a; Maddison, 1990,2000; Pagel,

1994,1997,2000,2002; Martins, 2000; Maddison & Mad-

dison, 2003; Pagel et al., 2004), which allow statistically

rigorous testing of correlations among characters, help us

to gain insight into which morphological characters may

be adaptive in differing habitats. Few studies of dipteran

taxa have used phylogenetic comparative methods to

study morphological adaptations that occurred in concert

with or in response to habitat transitions (e.g. Scheffer &

Wiegmann, 2000). Fewer yet have attempted to unravel

the morphological adaptations that have facilitated or

accompanied transitions between aquatic and terrestrial

habitats. Although Vermeij & Dudley (2000) reported

that transitions between aquatic and terrestrial habitats

are rare in plants and animals (with the exception of

tetrapod vertebrates), there are a number of dipteran

families that have sublineages that must have made such

transitions (e.g. Chironomidae, Dolichopodidae, Empid-

idae, Ephydridae, Muscidae, Sarcophagidae, Sciomyzi-

dae, Stratiomyidae, Syrphidae, Tabanidae, and

Tipulidae). Our study, which used phylogenetic compar-

ative methods to explore morphological adaptations to

both aquatic and terrestrial habitats in the sciomyzid

genus Tetanocera, is one of the first to do so within a

dipteran lineage.

The genus Tetanocera (Diptera: Sciomyzidae: Tetanoce-

rini) displays significant potential for studying morpho-

logical adaptations that may have occurred in concert

with or in response to habitat transitions for two reasons:

Correspondence: E. G. Chapman, Department of Biological Sciences,

Cunningham Hall, Kent State University, Kent, OH 44242-0001, USA.

Tel.: (330) 672-2921; fax: (330) 672-3713;

e-mail: echapman@kent.edu

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Keywords:

parallel evolution;

phylogenetic comparative method;

phylogenetic niche conservatism;

Sciomyzidae;

Tetanocera.

Abstract

In this study, we sequenced one nuclear and three mitochondrial DNA loci to

construct a robust estimate of phylogeny for all available species of Tetanocera.

Character optimizations suggested that aquatic habitat was the ancestral

condition for Tetanocera larvae, and that there were at least three parallel

transitions to terrestrial habitat, with one reversal. Maximum likelihood

analyses of character state transformations showed significant correlations

between habitat transitions and changes in four larval morphological charac-

teristics (cuticular pigmentation and three characters associated with the

posterior spiracular disc). We provide evidence that phylogenetic niche

conservatism has been responsible for the maintenance of aquatic-associated

larval morphological character states, and that concerted convergence and/or

gene linkage was responsible for parallel morphological changes that were

derived in conjunction with habitat transitions. These habitat–morphology

associations were consistent with the action of natural selection in facilitating

the morphological changes that occurred during parallel aquatic to terrestrial

habitat transitions in Tetanocera.

doi:10.1111/j.1420-9101.2006.01132.x

(i) larvae of its ca. 39 currently recognized species inhabit

both aquatic and terrestrial habitats (Foote,

1996a,b,1999); (ii) Larvae of aquatic species share a suite

of morphological characteristics that differentiates them

from the terrestrial species (Fig. 1). Aquatic Tetanocera

larvae live just under the surface of the water, usually

against dark floating debris or plant stems (Foote, 1999),

whereas terrestrial Tetanocera larvae occupy habitats

ranging from damp near-shore to drier woodland habi-

tats (Foote, 1996a,b). Aquatic Tetanocera larvae are darkly

pigmented (nearly black), which is likely for crypsis. The

posterior spiracular disc is typically modified in three

ways: (i) the last abdominal segment is lengthened and

the disc is upturned; (ii) the spiracles have a ring of long,

branched, hydrophobic hairs (float hairs); and (iii) there

are elongated, hirsute ventrolateral and ventral lobes

extending from the disc (Fig. 1a, b). The upturned

spiracular disc enables the larvae to breathe while

remaining ca. horizontal under the surface of the water.

The float hairs perform two functions: Aquatic Tetanocera

larvae attack and eat aquatic snails just below the surface

of the water (Foote, 1999). When a prey item dies or

flees, it sometimes loses contact with the surface of the

water, and drops through the water column, pulling the

feeding Tetanocera larva down with it. When this hap-

pens, the float hairs fold over the spiracles, trapping a

bubble of air (Foote, 1999). As the larva contacts the

surface, the hydrophobic float hairs break the surface

tension of the water and hold the spiracles (which

protrude slightly from the spiracular disc) above the

surface (Foote, 1999). The elongated hairy lobes around

the spiracular disc likely aid in keeping the disc at the

surface (B. A. Foote, unpublished data). In contrast,

terrestrial Tetanocera larvae are translucent (the nonpig-

mented cuticle is clear; the larvae appear white to tan).

The last abdominal segment is not lengthened and

terminates in a rear-facing posterior spiracular disc, and

the lobes and float hairs are greatly reduced (Fig. 1c, d).

The distribution of these two distinct suites of larval

character states, in multiple Tetanocera species, could be

the result of either a single habitat shift and subsequent

speciation or multiple shifts and parallel evolution in

larval morphology.

There are a number of interesting evolutionary ques-

tions one can ask about Tetanocera. Do the aquatic and

terrestrial species each comprise distinct lineages, or were

there multiple independent habitat transitions? The

answer to this question would allow us to address

whether the aforementioned morphological differences

between aquatic and terrestrial larvae were the result of a

single habitat transition and subsequent speciation, or if

multiple independent habitat transitions were each

accompanied by the same morphological changes as a

result of parallel evolution. If the latter were true, it

would be consistent with the hypothesis that adaptation

played a significant role in the transitions. Another

evolutionary question deals with the polarity and order

of larval character state transitions that occurred during

Tetanocera phylogenesis: what was the ancestral larval

habitat of Tetanocera? Did the lineage originate with

aquatic larvae, subsequently transitioning to terrestrial

habitats, or the reverse scenario? The nearly equal

number of aquatic and terrestrial species (of the

28 species with known life cycles, 13 are aquatic, 14 are

terrestrial and one is facultative) indicates that either

hypothesis should be considered plausible until they can

be evaluated using robust phylogenetic methods. This

study has four principal objectives: (i) use DNA sequences

to construct a robust estimate of phylogeny for all

available species of Tetanocera, (ii) estimate the polarity

and order of evolutionary transitions in larval habitat that

have occurred during Tetanocera phylogenesis, (iii) test

whether there is a significant correlation between larval

habitat and morphology and (iv) evaluate the hypothesis

that parallel habitat transitions were accompanied by

parallel state shifts in four larval morphological charac-

ters. Phylogenetic comparative methods have allowed us

to detect evolutionary phenomena such as parallel

evolution, concerted convergence/gene linkage, and

phylogenetic niche conservatism within Tetanocera.

Fig. 1 Illustrations of third instar Tetanocera

larvae: (a) Lateral view of Tetanocera ferrugi-

nea (aquatic larva); (b) Posterior spiracular

disc (rear view) of T. ferruginea; (c) lateral

view of T. melanostigma (terrestrial larva);

(d) posterior spiracular disc (rear view) of

T. melanostigma. Figures from Foote (1961).

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Materials and methods

Taxon sampling

The Sciomyzidae contains two subfamilies, with over

99% of the >500 species belonging to the Sciomyzinae

(Marinoni & Mathis, 2000; Knutson & Vala, 2002). The

Sciomyzinae is comprised of two tribes (Sciomyzini and

Tetanocerini). All of the Sciomyzini have terrestrial

larvae, whereas 14 tetanocerine genera have aquatic

larvae. A recent phylogenetic analysis of sciomyzid

morphological data suggests that the Sciomyzinae and

associated tribes are monophyletic (Marinoni & Mathis,

2000). Therefore, phylogenetic analyses were per-

formed on DNA sequences from four loci obtained

from 31 Tetanocera individuals (representing 17 species)

and 23 individuals representing eight outgroup genera

(six from the Tetanocerini and two from the Sciomy-

zini; Table 1). For 11 of the 17 Tetanocera species,

multiple individuals were available and sequenced for

replicate sampling purposes. In all but one case (the

Sciomyza simplex terminal), the four DNA loci sequenced

used to represent a particular terminal taxon were

obtained from the same individual. However, one

individual of S. simplex provided the three mtDNA-

encoded DNA sequences while another contributed the

28S sequence.

Tetanocera larvae feed on (i) nonoperculate pulmonate

aquatic snails in the water, (ii) nonoperculate pulmonate

aquatic snails on the shoreline, (iii) slugs (genera:

Deroceras, Pallifera, Philomycus), (iv) semi-terrestrial snails

in the family Succineidae and (v) terrestrial snails

(Knutson & Vala, 2002). Only those species that belong

to group no. 1 (above) have truly aquatic larvae. The

genus is Holarctic in distribution with 18 Nearctic, nine

Palaearctic and 12 Holarctic species. Eight of the

Tetanocera species analysed herein have aquatic larvae,

and nine have terrestrial larvae (two in group no. 2,

three in group no. 3, two in group no. 4, and two in

group no. 5). Together, these 17 species span the range of

morphological variation among known Tetanocera larvae

for the characters listed above.

Table 1 List of species analysed in this study with GenBank accession numbers and collecting locality information.

Tribe Species Habitat 28S 16S COI COII Locality

Tetanocerini Tetanocera bergi Steyskal, 1954 A AY875135 AY875104 AY875166 AY875197 USA: AK: Kanai

Tetanocera ferruginea Fallen, 1820 A AY875137 AY875106 AY875168 AY875199 USA: OH: Portage County

Tetanocera latifibula Frey, 1924 A AY875140 AY875109 AY875171 AY875202 Canada: MB: Churchill

Tetanocera mesopora Steyskal, 1959 A AY875142 AY875111 AY875173 AY875204 USA: ID: Buffalo River

Tetanocera montana Day, 1881 A AY875143 AY875112 AY875174 AY875205 Canada: AB; Banff NP

Tetanocera plumosa Loew, 1847 A AY875146 AY875115 AY875177 AY875208 USA: CO: Teller County

Tetanocera robusta Loew, 1847 A AY875147 AY875116 AY875178 AY875209 USA: CO: Teller County

Tetanocera vicina Macquart, 1843 A AY875150 AY875119 AY875181 AY875212 USA: OH: Geauga County

Tetanocera fuscinervis (Zetterstedt, 1838) T AY875138 AY875107 AY875169 AY875200 USA: CO: Teller County

Tetanocera silvatica Meigen, 1830 T AY875148 AY875117 AY875179 AY875210 Germany

Tetanocera kerteszi Hendel 1901 T AY875139 AY875108 AY875170 AY875201 USA: CO: Teller County

Tetanocera phyllophora Melander, 1920 T AY875144 AY875113 AY875175 AY875206 Germany: Ludvika

Tetanocera clara Loew, 1862 T AY875136 AY875105 AY875167 AY875198 USA: OH: Muskingum County

Tetanocera plebeja Loew, 1862 T AY875145 AY875114 AY875176 AY875207 USA: CO: Teller County

Tetanocera valida Loew, 1862 T AY875149 AY875118 AY875180 AY875211 USA: WV: Tucker County

Tetanocera arrogans Meigen, 1830 T AY875134 AY875103 AY875165 AY875196 Ireland

Tetanocera melanostigma Steyskal, 1959 T AY875141 AY875110 AY875172 AY875203 USA

Elgiva connexa Steyskal, 1954 A AY875122 AY875091 AY875153 AY875184 Canada: MB: Churchill

Elgiva solicita (Harris, 1780) A AY875123 AY875092 AY875154 AY875185 USA: OH

Hedria mixta Steyskal, 1954 A AY875124 AY875093 AY875155 AY875186 Canada: MB: Churchill

Limnia bosci Robineau-Desvoidy, 1830 T AY875125 AY875094 AY875156 AY875187 USA: MN: Cass County

Limnia ottawensis Melander, 1920 ? AY875126 AY875095 AY875157 AY875188 USA: SD: Custer Co. Custer SP

Limnia sandovalensis Fisher & Orth, 1978 ? AY875127 AY875096 AY875158 AY875189 Canada: AB; Banff NP

Renocera amanda (Cresson 1920) A AY875128 AY875097 AY875159 AY875190 USA: OH: Summit County

Renocera johnsoni (Cresson, 1920) A AY875129 AY875098 AY875160 AY875191 USA: CO: Teller County

Sepedon armipes Loew, 1859 A AY875130 AY875099 AY875161 AY875192 USA: OH: Portage County

Sepedon fuscipennis Loew, 1859 A AY875131 AY875100 AY875162 AY875193 USA: ND: Bottineau County

Sepedon praemiosa Giglio-Tos, 1893 A AY875132 AY875101 AY875163 AY875194 USA: ID: Bear Lake County

Trypetoptera canadensis (Macquart, 1843) T AY875133 AY875102 AY875164 AY875195 Canada: SK: Maple Creek

Sciomyzini Atrichomelina pubera (Loew, 1862) T AY875120 AY875089 AY875151 AY875182 Canada: SK: Cypress Hills

Sciomyza simplex Fallen, 1820 T AY875121 AY875090 AY875152 AY875183 Canada: AB: Banff NP

Habitat designations: A, aquatic; T, terrestrial. Park abbreviations: NP, National Park; SP, State Park.

Parentheses around the author and year indicates that the species was described under a different genus name. No parentheses indicates that

the species was described under the current genus.

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Laboratory protocols

Field collections of adult specimens were preserved

immediately in 95–100% nondenatured ethanol. In the

lab, specimens were transferred to vials containing 100%

hexamethyldisilazane (HMDS) for at least 24 h, after

which the liquid was decanted, and the specimens were

allowed to dry under a hood. In preparation for total

DNA isolation, the head, legs, wings, and abdomen of

each specimen were removed from the thorax. Total

DNA was isolated from each thorax, and the remaining

body parts (which contain the morphological characters

necessary for species determination) were stored as

vouchers in a vial containing 95–100% ethanol. Each

specimen was given a unique number, and species

identification, collecting locality information, and habitat

notes were recorded in a database.

Total DNA was isolated, using Qiagen DNeasy Tissue

Kits, from each of the terminal taxa (¼sciomyzid species)

in the analyses. Each of the DNA isolates was PCR-

amplified using the primer pairs listed in Table 2. The

mitochondrial and nuclear amplicons were characterized

by cycle sequencing analysis using the PCR amplification

primers listed in Table 2. The protocols for sequencing

template purification and cycle sequencing of the frag-

ments are as presented in Folmer et al. (1994). These

protocols include sequencing template purification in

low-melting point agarose gels and cycle sequencing of

both strands of each purified template using labelled

primers. The separation of cycle sequencing reaction

products was done in 3.7% and 5.5% polyacrylamide

gels on LI-COR 4200L-2 and 4200S-2 automated DNA

sequencers respectively. The resulting sequences were

aligned initially using ALIGNIRALIGNIR (ALIGNIR V2.0ALIGNIR V2.0, LI-COR

Inc.) with subsequent refinement done manually using

MACCLADE V. 4.0MACCLADE V. 4.0 (Maddison & Maddison, 2000). All

sequences presented in this study have been deposited in

the GenBank database (see Table 1 for accession num-

bers). The alignment of the COI and COII sequences were

straightforward, as no indels have been detected in the

sciomyzid sequences generated to date from these loci.

While the 28S sequences contain some indels, we have

found no regions that align ambiguously. However,

indels that do present alignment ambiguities have been

detected in the sciomyzid 16S sequences. Therefore, to

avoid arbitrarily derived topologies, phylogenetic analy-

ses were carried out with both (i) a ‘best’ alignment

containing all of the 16S nucleotides, and (ii) an

alignment with the ambiguous 16S characters deleted.

Phylogenetic analyses

The mtDNA-encoded COI, COII, 16S and the nucleus-

encoded 28S sequences (nuDNA) were analysed using

the maximum likelihood (ML) and maximum parsimony

(MP) algorithms contained in PAUPPAUP* (v.4.0b10; Swofford,

2001). Bayesian inference (BI) analyses were carried out

with MRBAYES V3.1MRBAYES V3.1 (Huelsenbeck & Ronquist, 2001;

Ronquist & Huelsenbeck, 2003). The parsimony-based

ILD test (Farris et al., 1994), as implemented in PAUPPAUP*,

was used to test for incongruence between the mtDNA

and nuDNA datasets. The COI, COII, 16S, and 28S

sequences were analysed simultaneously, as recent

literature indicates that a total evidence approach can

produce the best tree topologies (Collin, 2003; Creer

et al., 2003; Hassanin & Douzery, 2003; Schwarz et al.,

2003). Thus, the total evidence-based trees were used as

the best estimates of the phylogenetic relationships

among Tetanocera species. MODELTESTMODELTEST (V. 3.6:V. 3.6: Posada &

Crandall, 1998) was used to determine which model best

fit the concatenated sequence data. The GTR + G + I

model was used in all BI and ML analyses. Atrichomelina

pubera (Sciomyzini) sequences were used to root the

trees. The ML algorithm in PAUPPAUP*, using the parameters

from the output of MODELTESTMODELTEST, was implemented to

judge which of the 1001 trees sampled from the Bayesian

analysis had the highest log likelihood value and, thus,

represented the best topology.

A total of 54 specimens, representing 31 species, were

included in the initial BI analysis (four chains, two

million generations, 50 000 generation burn-in,

GTR + I + G), after which duplicate individuals for each

Table 2 Genes/primer information used in

this study.Gene primer pair References

Amplicon

size (bp) Notes

Mitochondrial loci

16S LR-N-13398/LR-J-12887 Simon et al. (1994) �550 Primer sequences identical to

those of ‘Locust’

COI LCO1490/HCO2198 Folmer et al. (1994) �700 Together, both COI

COI C1-J-2183/TL2-N-3014 Simon et al. (1994) �800 primer pairs encompass nearly

the entire gene

COII TL2-J-3034/TK-N-3785 Simon et al. (1994)

J.B. Hobbs, UBC

(personal communication)

�800 Amplifies all of COII

Nuclear locus

28S D1F/D6R Park & O’Foighil (2000) �1100

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species were pruned (as all conspecific individuals

formed monophyletic groups with 100% posterior prob-

abilities). Four independent Bayesian analyses were

performed to explore whether they all would arrive at

the same topology. Subsequent MP and ML analyses

were carried out with the pruned dataset, to investigate

whether the use of other tree building algorithms

resulted in congruent or conflicting topologies. Multiple

random terminal taxa addition sequence runs, combined

with global branch rearrangement options, were

employed when generating topologies from the ML and

MP algorithms. These options increased the probability of

finding the actual best topology under each of these two

optimality criteria (e.g. Hendy et al., 1988; Maddison,

1991). Standard bootstrap (Felsenstein, 1985b) analyses

were carried out to evaluate the level of support for

particular nodes obtained from the ML (100 bootstrap

replicates) and MP (10 000 bootstrap replicates) analyses.

Pairwise uncorrected p-distances were calculated, using

PAUPPAUP*, for each gene. To test for significant differences in

topologies between the best unconstrained tree and a

topology produced by constraining the terrestrial Tetano-

cera species to be monophyletic, we used (i) PAUPPAUP* to do

the parsimony-based Kishino-Hasegawa test (KH;

Kishino & Hasegawa, 1989), Templeton test (Wilcoxon

signed-ranks test; Templeton, 1983) and winning sites

(sign) test (Prager & Wilson, 1988) and (ii) CONSEL

(Shimodaira & Hasegawa, 2001) to do the likelihood-

based approximately unbiased (AU, Shimodaira, 2002),

Kishino-Hasegawa (KH), Shimodaira-Hasegawa (SH;

Shimodaira & Hasegawa, 1999), weighted Kishino-

Hasegawa (WKH), and weighted Shimodaira–Hasegawa

(WSH; Shimodaira, 2002) tests. The estimation of ances-

tral character states, based on our best estimate of

phylogeny, was carried out using equally weighted

parsimony methods (e.g. Scheffer & Wiegmann, 2000;

Jousselin et al., 2003; Pauly et al., 2004) and with ML

methods, both using MESQUITEMESQUITE (V.1.05V.1.05; Maddison &

Maddison, 2003). Although Tetanocera plumosa can be

found in both aquatic and wet shoreline (terrestrial)

habitats, it is principally aquatic (Foote, 1961) and was

scored as such in all character optimization procedures.

For the ML optimizations, both the ‘Markov k-state 1

parameter model’ (MK1 model in which ‘forward’ and

‘backward’ transition rates are equal) and the ‘Asym-

metrical Markov k-state 2 parameter model’ (AsymmMK

model in which ‘forward’ and ‘backward’ transition rates

can be different) were used. The asymmetry likelihood

ratio test was used to determine whether the AsymmMK

model was significantly better than the MK1 model.

Tests of correlated evolution

The maximum-likelihood program DISCRETEDISCRETE (Pagel,

1994,1997,1999a,b) was used to test for correlated

evolution between ecological and morphological charac-

ters (omnibus test). This test utilizes a Markov model in a

ML framework, taking branch length information into

account, but does not rely on ancestral character state

reconstruction. Given a pair of binary characters and a

tree topology, the program calculates the log-likelihoods

for two models: (i) a model in which the two characters

are allowed to evolve independently (independent

model) and (ii) a model in which the two characters

evolve in a correlated manner (dependent model: Fig. 2).

A Monte Carlo simulation study, in which character

states are repeatedly assigned independently and ran-

domly to the terminals of the tree, approximates the null

hypothesis distribution for the characters at hand. The

outcome of the simulation is used to determine whether

the independent or dependent model of character evo-

lution best fits the data, via a likelihood ratio test. If the

dependent model (correlated evolution model) fits the

data significantly better than the independent model,

then the null hypothesis that the two traits evolved

independently is rejected. The omnibus test was also used

to test for correlated evolution among morphological

characters. When evidence for correlated evolution was

found, DISCRETEDISCRETE was used to test whether a given trait

changes from state 0 to state 1 before the other (temporal

order test). This is also tested via a likelihood ratio test

that compares the log-likelihood of the full eight-

parameter (dependent) model to that of a seven-param-

eter model in which q12 and q13 are set to be equal to one

another (see Fig. 2). In other words, if two times the

difference in log-likelihoods between the two models is

larger than 3.84 (v2 value with one degree of freedom;

a ¼ 0.05), then the null hypothesis that neither charac-

ter tends to change before the other can be rejected.

Rejecting the null hypothesis of the omnibus test,

combined with failure to reject the null hypothesis of

Fig. 2 Flow diagram showing how habitat [aquatic (AQ) and

terrestrial (T)] and a given morphological character [morphology

typical of aquatic species (Maq) and morphology typical of terrestrial

species (MT)] may evolve in a correlated fashion. The eight qij values

are ‘forward’ and ‘backward’ transition rate parameters estimated

from the data. It is assumed that both traits do not change at the

same time (i.e. q14, q41, q23, and q32 all equal zero).

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the temporal order test indicates that while the two traits

are evolving in a correlated fashion, it is not possible to

tell which trait changes first. When two morphological

traits are being tested, this result indicates that the two

traits are linked by (i) pleiotropy, (ii) indirect selection

through another trait or (iii) concerted evolution

(Armbruster, 2002), but other methods are necessary to

distinguish between these.

We tested whether there were significant correlations

among all possible combinations of larval habitat and the

four morphological characters outlined above. These tests

were conducted on both the entire phylogeny and the

Tetanocera portion only. As Tetanocera plumosa can be

found in both habitats, and the spiracular disc orientation

of T. silvatica could not be determined, all possible

scenarios involving these taxa were tested. All variables

were scored as discrete binary characters as there is a

clear distinction between each scored character’s char-

acter states; [coding: larvae with pigmented cuticle ¼ 0,

unpigmented larvae ¼ 1; float hairs long (extending well

beyond the base of the spiracular tube), branched,

conspicuous ¼ 0, short (not or barely extending beyond

the base of the spiracular tube), unbranched ¼ 1; ven-

trolateral lobes longer than width at base ¼ 0, shorter/

equal to basal width ¼ 1; posterior spiracular disc

upturned ¼ 0, posterior spiracular disc not upturned ¼1]. Larval character states and habitat designations were

obtained from Foote (1959,1961,1971,1976, unpublished

data), Foote et al. (1960), Knutson (1963), Knutson &

Berg (1964), Neff & Berg (1966), and Knutson & Vala

(2002). The sequential Bonferroni technique (Rice,

1989) was used to minimize type I statistical error, the

likelihood of which is increased when performing multi-

ple statistical tests (also see Holm, 1979).

Results

Phylogenetic analyses

Pairwise uncorrected p-distances for 16S and 28S rDNA

sequences are given in Table 3, and those for COI and

COII are given in Table 4. The tree topologies obtained

from the three phylogenetic analyses of the concatenated

dataset were largely congruent, with the BI tree fully

resolved (Fig. 3) and the MP and ML bootstrap trees (not

shown) recovering the same major clades as indicated in

the BI analysis (albeit with lower intra-clade resolution).

All four independent BI analyses converged on the same

topology. The best BI (as judged by ML) and ML trees

were identical (Ln L ¼ )37275.65). The MP analysis of

1217 parsimony-informative characters in the concaten-

ated dataset, produced two equally parsimonious trees

(Ln L ¼ )37322.21 and )37323.07 as judged by ML)

which had identical topologies within Tetanocera, only

differing from the BI and ML trees in the placement of

T. latifibula and T. mesopora: In the MP trees, T. latifibula is

sister to T. montana + T. kerteszi, and T. mesopora is sister to

a clade comprised of the three aforementioned species,

whereas T. latifibula and T. mesopora are sister taxa on the

BI and ML trees. As T. kerteszi is the only terrestrial

species among these four (L.V. Knutson, personal com-

munication), either topology would give nearly identical

results with respect to both character optimizations and

correlated evolution tests. Concatenating the nuDNA and

mtDNA sequences was legitimized by the lack of signi-

ficant incongruence between the datasets (as indicated

by the ILD test, P ¼ 0.974). In the BI analysis, of the

29 internal nodes, 23 were supported by posterior

probabilities ‡0.90, three were between 0.80 and 0.89,

and there was one each in the 0.70, 0.60 and 0.50s

(lowest pp ¼ 0.59).

Tetanocera is clearly supported as a monophyletic group

based on very high nodal support values (BI posterior

probability ¼ 1.00; ML bootstrap percentage ¼ 100; MP

bootstrap percentage ¼ 90; Fig. 3). Within Tetanocera,

three well-supported subclades were inferred, each con-

taining at least one aquatic and one terrestrial species

(Fig. 3 and Table 1). The robusta-silvatica clade (BI pp ¼1.00, ML bootstrap percentage ¼ 100, MP bootstrap

percentage ¼ 100) is sister to the remaining Tetanocera

species. It contains two species: one aquatic and one

terrestrial. The plumosa-mesopora clade (BI pp ¼ 1.00, ML

bootstrap percentage ¼ 82, MP bootstrap percentage ¼65) contains four aquatic, one terrestrial, and one species

(T. plumosa) that can be found both in water and on

shorelines (Foote, 1961). The phyllophora-valida clade (BI

pp ¼ 1.00, ML bootstrap percentage ¼ 97, MP bootstrap

percentage ¼ 74) contains two aquatic and seven ter-

restrial species.

Larval habitats (aquatic vs. terrestrial) of the sciomyzid

species were mapped onto the BI topology using ML

(Fig. 4) and parsimony (Fig. 5) methods. The asymmetry

likelihood ratio test showed that the AsymmMK model

was not significantly better than the MK1 model. Of the

25 internal nodes on the tree, 19 had ancestral states that

were statistically significant as judged by the AsymmMk

optimization model (nodes with an asterisk on Fig. 4a),

and 13 had ancestral states that were statistically signifi-

cant as judged by the MK1 optimization model (nodes

with an asterisk on Fig. 4b). From this analysis, it can be

inferred that the ancestral larvae of Tetanocera was very

likely aquatic (AsymmMk ML estimate ¼ 0.97; MK1 ML

estimate ¼ 0.82). From this ancestral condition, there

were at least three independent transitions to terrestrial

existence, with one reversal (also see parsimony optimi-

zation in Fig. 5). Since the evidence supporting a terrest-

rial ancestral state for the phyllophora-valida clade is

relatively weak (AsymmMk ML estimate ¼ 0.54; MK1

ML estimate ¼ 0.64) as it is for the subsequent node after

T. phyllophora arose (AsymmMk ML estimate ¼ 0.62;

MK1 ML estimate ¼ 0.72), it is possible that the ancestor

of the phyllophora-valida clade was, in fact, aquatic. If this

were true, then terrestrial existence arose five times

independently within Tetanocera, with no reversals.

1464 E. G. CHAPMAN ET AL.

ª 2 0 0 6 T H E A U T H O R S 1 9 ( 2 0 0 6 ) 1 4 5 9 – 1 4 7 4

J O U R N A L C O M P I L A T I O N ª 2 0 0 6 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y

Tab

le3

Pair

-wis

eu

nco

rrect

ed

p-d

ista

nce

sbetw

een

scio

myzi

dsp

eci

es

appeari

ng

inTable

1.

Sp

ecie

s1

23

45

67

89

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

1.

Atr

ichom

elin

ap

ub

era

–0.0

20

0.0

52

0.0

59

0.0

54

0.0

54

0.0

58

0.0

54

0.0

65

0.0

63

0.0

57

0.0

54

0.0

56

0.0

51

0.0

54

0.0

53

0.0

55

0.0

50

0.0

53

0.0

52

0.0

48

0.0

57

0.0

47

0.0

54

0.0

59

0.0

51

0.0

48

0.0

52

0.0

56

0.0

60

0.0

49

2.

Scio

myz

asi

mp

lex

0.0

33

–0.0

46

0.0

54

0.0

50

0.0

48

0.0

57

0.0

48

0.0

62

0.0

55

0.0

53

0.0

53

0.0

50

0.0

48

0.0

51

0.0

49

0.0

53

0.0

45

0.0

50

0.0

50

0.0

45

0.0

54

0.0

44

0.0

52

0.0

55

0.0

50

0.0

44

0.0

49

0.0

51

0.0

54

0.0

45

3.

Elg

iva

connexa

0.0

96

0.1

04

–0.0

20

0.0

20

0.0

22

0.0

25

0.0

22

0.0

40

0.0

36

0.0

32

0.0

30

0.0

29

0.0

19

0.0

20

0.0

20

0.0

22

0.0

16

0.0

19

0.0

21

0.0

20

0.0

28

0.0

18

0.0

24

0.0

25

0.0

19

0.0

15

0.0

24

0.0

24

0.0

27

0.0

18

4.

Elg

iva

solic

ita0.0

98

0.1

00

0.1

10

–0.0

32

0.0

36

0.0

38

0.0

36

0.0

51

0.0

48

0.0

37

0.0

38

0.0

36

0.0

33

0.0

32

0.0

30

0.0

36

0.0

27

0.0

29

0.0

32

0.0

31

0.0

39

0.0

30

0.0

35

0.0

36

0.0

31

0.0

28

0.0

34

0.0

35

0.0

40

0.0

31

5.

Hed

ria

mix

ta0.0

90

0.0

78

0.1

07

0.0

96

–0.0

09

0.0

21

0.0

09

0.0

39

0.0

33

0.0

35

0.0

32

0.0

32

0.0

07

0.0

20

0.0

18

0.0

21

0.0

14

0.0

15

0.0

21

0.0

18

0.0

26

0.0

15

0.0

24

0.0

23

0.0

19

0.0

15

0.0

23

0.0

22

0.0

26

0.0

18

6.

Lim

nia

bosc

i0.1

08

0.1

10

0.1

33

0.1

13

0.0

86

–0.0

25

0.0

00

0.0

39

0.0

30

0.0

33

0.0

31

0.0

32

0.0

05

0.0

23

0.0

22

0.0

25

0.0

18

0.0

19

0.0

23

0.0

20

0.0

29

0.0

18

0.0

26

0.0

26

0.0

23

0.0

17

0.0

25

0.0

19

0.0

27

0.0

20

7.

Lim

nia

ott

aw

ensi

s0.1

32

0.1

18

0.1

37

0.1

27

0.0

93

0.1

06

–0.0

25

0.0

49

0.0

42

0.0

41

0.0

37

0.0

38

0.0

23

0.0

18

0.0

16

0.0

19

0.0

15

0.0

15

0.0

25

0.0

19

0.0

25

0.0

17

0.0

27

0.0

19

0.0

16

0.0

17

0.0

19

0.0

23

0.0

26

0.0

19

8.

Lim

nia

sand

ova

lensi

s0.1

08

0.1

10

0.1

33

0.1

13

0.0

86

0.0

00

0.1

06

–0.0

39

0.0

30

0.0

33

0.0

31

0.0

32

0.0

05

0.0

23

0.0

22

0.0

25

0.0

18

0.0

19

0.0

23

0.0

20

0.0

29

0.0

18

0.0

26

0.0

26

0.0

23

0.0

17

0.0

25

0.0

19

0.0

27

0.0

20

9.

Renocera

am

and

a0.1

17

0.1

05

0.1

14

0.1

18

0.1

02

0.1

14

0.1

36

0.1

14

–0.0

43

0.0

49

0.0

41

0.0

47

0.0

37

0.0

37

0.0

39

0.0

38

0.0

35

0.0

37

0.0

37

0.0

37

0.0

45

0.0

35

0.0

40

0.0

42

0.0

36

0.0

32

0.0

40

0.0

40

0.0

45

0.0

35

10.

Renocera

johnso

ni

0.0

92

0.0

96

0.1

31

0.1

14

0.1

18

0.1

12

0.1

22

0.1

12

0.1

24

–0.0

43

0.0

46

0.0

44

0.0

28

0.0

32

0.0

31

0.0

34

0.0

29

0.0

31

0.0

32

0.0

30

0.0

38

0.0

28

0.0

35

0.0

36

0.0

34

0.0

27

0.0

33

0.0

27

0.0

40

0.0

29

11.

Sep

ed

on

arm

ipes

0.1

28

0.1

26

0.1

32

0.1

36

0.1

35

0.1

58

0.1

43

0.1

58

0.1

60

0.1

35

–0.0

24

0.0

04

0.0

30

0.0

29

0.0

32

0.0

34

0.0

30

0.0

31

0.0

34

0.0

32

0.0

34

0.0

30

0.0

37

0.0

36

0.0

28

0.0

28

0.0

35

0.0

34

0.0

38

0.0

31

12.

Sep

ed

on

fusc

ipennis

0.1

25

0.1

33

0.1

35

0.1

28

0.1

36

0.1

39

0.1

61

0.1

39

0.1

63

0.1

41

0.1

04

–0.0

21

0.0

29

0.0

27

0.0

30

0.0

32

0.0

27

0.0

29

0.0

29

0.0

29

0.0

31

0.0

27

0.0

32

0.0

32

0.0

24

0.0

24

0.0

31

0.0

30

0.0

35

0.0

25

13.

Sep

ed

on

pra

em

iosa

0.1

34

0.1

32

0.1

34

0.1

48

0.1

37

0.1

68

0.1

45

0.1

68

0.1

72

0.1

43

0.0

91

0.0

94

–0.0

30

0.0

26

0.0

30

0.0

31

0.0

27

0.0

30

0.0

32

0.0

29

0.0

32

0.0

27

0.0

34

0.0

34

0.0

25

0.0

24

0.0

33

0.0

33

0.0

37

0.0

27

14.

Try

peto

pte

racanad

ensi

s0.1

32

0.1

22

0.1

45

0.1

22

0.1

07

0.1

18

0.0

83

0.1

18

0.1

28

0.1

33

0.1

46

0.1

45

0.1

33

–0.0

20

0.0

19

0.0

22

0.0

15

0.0

16

0.0

20

0.0

17

0.0

27

0.0

15

0.0

23

0.0

23

0.0

20

0.0

14

0.0

22

0.0

17

0.0

25

0.0

17

15.

Try

peto

pte

raarr

ogans

0.1

38

0.1

36

0.1

69

0.1

56

0.1

47

0.1

54

0.1

74

0.1

54

0.1

64

0.1

46

0.1

80

0.1

76

0.1

85

0.1

61

–0.0

10

0.0

11

0.0

07

0.0

11

0.0

15

0.0

12

0.0

13

0.0

10

0.0

17

0.0

13

0.0

08

0.0

08

0.0

15

0.0

18

0.0

17

0.0

10

16.

Try

peto

pte

rab

erg

i0.1

12

0.1

26

0.1

36

0.1

42

0.1

44

0.1

50

0.1

69

0.1

50

0.1

50

0.1

43

0.1

63

0.1

52

0.1

56

0.1

51

0.1

27

–0.0

14

0.0

04

0.0

10

0.0

16

0.0

11

0.0

18

0.0

10

0.0

19

0.0

12

0.0

10

0.0

08

0.0

12

0.0

13

0.0

18

0.0

10

17.

Try

peto

pte

racla

ra0.1

38

0.1

38

0.1

56

0.1

60

0.1

49

0.1

47

0.1

61

0.1

47

0.1

52

0.1

45

0.1

88

0.1

66

0.1

79

0.1

47

0.1

12

0.1

16

–0.0

10

0.0

14

0.0

21

0.0

16

0.0

15

0.0

14

0.0

24

0.0

17

0.0

11

0.0

12

0.0

18

0.0

20

0.0

13

0.0

15

18.

Try

peto

pte

rafe

rrugin

ea

0.1

12

0.1

26

0.1

36

0.1

42

0.1

44

0.1

50

0.1

69

0.1

50

0.1

48

0.1

43

0.1

61

0.1

52

0.1

54

0.1

51

0.1

29

0.0

02

0.1

16

–0.0

07

0.0

12

0.0

08

0.0

14

0.0

06

0.0

15

0.0

10

0.0

07

0.0

04

0.0

10

0.0

11

0.0

14

0.0

07

19.

Teta

nocera

fusc

inerv

is0.1

30

0.1

38

0.1

34

0.1

59

0.1

72

0.1

62

0.1

77

0.1

62

0.1

59

0.1

53

0.1

71

0.1

56

0.1

64

0.1

75

0.1

51

0.1

08

0.1

20

0.1

06

–0.0

15

0.0

10

0.0

18

0.0

09

0.0

18

0.0

14

0.0

11

0.0

09

0.0

15

0.0

16

0.0

19

0.0

11

20.

Teta

nocera

kert

esz

i0.1

16

0.1

20

0.1

36

0.1

25

0.1

26

0.1

46

0.1

49

0.1

46

0.1

43

0.1

29

0.1

49

0.1

54

0.1

42

0.1

46

0.1

18

0.1

14

0.1

18

0.1

12

0.1

20

–0.0

09

0.0

24

0.0

07

0.0

03

0.0

20

0.0

13

0.0

09

0.0

18

0.0

18

0.0

25

0.0

10

21.

Teta

nocera

latifi

bula

0.1

33

0.1

29

0.1

40

0.1

45

0.1

42

0.1

52

0.1

62

0.1

52

0.1

45

0.1

34

0.1

63

0.1

64

0.1

64

0.1

54

0.1

33

0.1

22

0.1

31

0.1

20

0.1

22

0.0

85

–0.0

16

0.0

03

0.0

12

0.0

16

0.0

12

0.0

05

0.0

14

0.0

13

0.0

20

0.0

07

22.

Teta

nocera

mela

nost

igm

a0.1

39

0.1

42

0.1

43

0.1

61

0.1

59

0.1

65

0.1

72

0.1

65

0.1

62

0.1

59

0.1

98

0.1

95

0.1

91

0.1

76

0.1

61

0.1

38

0.1

45

0.1

39

0.1

33

0.1

37

0.1

41

–0.0

17

0.0

27

0.0

19

0.0

14

0.0

16

0.0

22

0.0

24

0.0

20

0.0

17

23.

Teta

nocera

meso

pora

0.1

39

0.1

39

0.1

39

0.1

55

0.1

40

0.1

51

0.1

68

0.1

51

0.1

35

0.1

30

0.1

60

0.1

53

0.1

46

0.1

48

0.1

30

0.1

15

0.1

15

0.1

14

0.1

17

0.0

81

0.0

89

0.1

43

–0.0

09

0.0

14

0.0

10

0.0

02

0.0

12

0.0

11

0.0

18

0.0

04

24.

Teta

nocera

monta

na

0.1

26

0.1

34

0.1

40

0.1

35

0.1

36

0.1

54

0.1

57

0.1

54

0.1

55

0.1

37

0.1

53

0.1

58

0.1

46

0.1

54

0.1

19

0.1

20

0.1

24

0.1

18

0.1

26

0.0

18

0.0

89

0.1

43

0.0

93

–0.0

23

0.0

16

0.0

11

0.0

21

0.0

21

0.0

28

0.0

13

25.

Teta

nocera

phyl

lop

hora

0.1

12

0.1

12

0.1

42

0.1

58

0.1

40

0.1

42

0.1

59

0.1

42

0.1

39

0.1

23

0.1

73

0.1

64

0.1

63

0.1

45

0.1

31

0.0

93

0.1

30

0.0

91

0.1

12

0.1

14

0.1

14

0.1

24

0.1

04

0.1

20

–0.0

11

0.0

13

0.0

15

0.0

19

0.0

20

0.0

16

26.

Teta

nocera

ple

beja

0.1

39

0.1

49

0.1

63

0.1

59

0.1

58

0.1

62

0.1

87

0.1

62

0.1

44

0.1

38

0.1

80

0.1

85

0.1

87

0.1

74

0.1

36

0.1

17

0.1

34

0.1

18

0.1

25

0.1

23

0.1

34

0.1

50

0.1

10

0.1

29

0.1

19

–0.0

09

0.0

14

0.0

17

0.0

17

0.0

11

27.

Teta

nocera

plu

mosa

0.1

51

0.1

47

0.1

49

0.1

49

0.1

48

0.1

45

0.1

72

0.1

45

0.1

41

0.1

56

0.1

71

0.1

72

0.1

68

0.1

64

0.1

53

0.1

44

0.1

31

0.1

42

0.1

50

0.1

07

0.1

25

0.1

62

0.1

05

0.1

13

0.1

40

0.1

42

–0.0

11

0.0

12

0.0

17

0.0

02

28.

Teta

nocera

rob

ust

a0.1

18

0.1

18

0.1

18

0.1

29

0.1

31

0.1

44

0.1

61

0.1

44

0.1

33

0.1

29

0.1

59

0.1

64

0.1

61

0.1

53

0.1

13

0.1

20

0.1

18

0.1

20

0.1

14

0.1

04

0.0

98

0.1

26

0.0

95

0.1

02

0.1

08

0.1

31

0.1

23

–0.0

17

0.0

21

0.0

14

29.

Teta

nocera

silv

atic

a0.1

47

0.1

35

0.1

37

0.1

42

0.1

45

0.1

59

0.1

68

0.1

59

0.1

39

0.1

58

0.1

58

0.1

76

0.1

85

0.1

64

0.1

57

0.1

38

0.1

56

0.1

36

0.1

42

0.1

18

0.1

23

0.1

56

0.1

09

0.1

32

0.1

30

0.1

44

0.1

39

0.1

14

–0.0

24

0.0

15

30.

Teta

nocera

valid

a0.1

35

0.1

31

0.1

57

0.1

60

0.1

53

0.1

61

0.1

74

0.1

61

0.1

54

0.1

31

0.1

71

0.1

71

0.1

70

0.1

65

0.1

32

0.1

11

0.1

04

0.1

11

0.1

24

0.1

08

0.0

92

0.1

43

0.1

02

0.1

16

0.1

03

0.1

19

0.1

36

0.1

17

0.1

38

–0.0

18

31.

Teta

nocera

vicin

a0.1

37

0.1

35

0.1

46

0.1

43

0.1

56

0.1

46

0.1

72

0.1

46

0.1

53

0.1

48

0.1

62

0.1

70

0.1

72

0.1

62

0.1

42

0.1

39

0.1

23

0.1

37

0.1

38

0.1

03

0.1

23

0.1

53

0.1

25

0.1

11

0.1

41

0.1

50

0.0

79

0.1

02

0.1

33

0.1

22

–

Low

er

left

,16S

rDN

Ap-d

ista

nce

s;u

pper

righ

t,28S

p-d

ista

nce

s.

Habitat correlated parallel evolution 1465

ª 2 0 0 6 T H E A U T H O R S 1 9 ( 2 0 0 6 ) 1 4 5 9 – 1 4 7 4

J O U R N A L C O M P I L A T I O N ª 2 0 0 6 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y

Tab

le4

Pair

wis

eu

nco

rrect

ed

p-d

ista

nce

sbetw

een

scio

myzi

dsp

eci

es

appeari

ng

inTable

1.

Sp

ecie

s1

23

45

67

89

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

1.

Atr

ichom

elin

ap

ub

era

–0.1

14

0.1

88

0.1

69

0.2

35

0.1

97

0.1

97

0.1

99

0.2

34

0.2

35

0.1

97

0.1

91

0.1

87

0.2

23

0.2

70

0.2

26

0.2

57

0.2

32

0.2

48

0.2

37

0.2

20

0.2

59

0.2

09

0.2

37

0.2

38

0.2

38

0.2

05

0.1

84

0.2

33

0.2

74

0.2

35

2.

Scio

myz

asi

mp

lex

0.1

22

–0.2

08

0.1

88

0.2

35

0.2

15

0.2

08

0.2

14

0.2

30

0.2

32

0.2

00

0.2

15

0.2

18

0.2

36

0.2

65

0.2

29

0.2

71

0.2

35

0.2

68

0.2

46

0.2

35

0.2

68

0.2

24

0.2

51

0.2

59

0.2

68

0.2

09

0.1

94

0.2

42

0.2

65

0.2

33

3.

Elg

iva

connexa

0.1

72

0.1

78

–0.1

82

0.2

02

0.1

96

0.2

02

0.1

94

0.2

36

0.2

23

0.2

08

0.2

14

0.1

81

0.2

06

0.2

35

0.2

20

0.2

18

0.2

18

0.2

26

0.2

07

0.1

97

0.2

60

0.1

85

0.2

34

0.2

31

0.2

40

0.1

96

0.1

99

0.2

11

0.2

44

0.2

24

4.

Elg

iva

solic

ita0.1

66

0.1

63

0.1

68

–0.2

00

0.2

02

0.1

82

0.2

03

0.2

38

0.2

21

0.1

81

0.1

72

0.1

79

0.1

84

0.2

52

0.2

15

0.2

51

0.2

20

0.2

27

0.2

29

0.2

03

0.2

44

0.2

00

0.2

22

0.2

29

0.2

49

0.1

96

0.1

82

0.2

26

0.2

44

0.2

18

5.

Hed

ria

mix

ta0.1

96

0.1

80

0.1

78

0.1

76

–0.2

11

0.1

90

0.2

09

0.2

63

0.2

33

0.2

12

0.2

27

0.2

21

0.1

84

0.2

59

0.2

36

0.2

62

0.2

30

0.2

68

0.2

43

0.2

36

0.2

59

0.2

30

0.2

59

0.2

49

0.2

61

0.2

30

0.2

12

0.2

56

0.2

53

0.2

30

6.

Lim

nia

bosc

i0.1

81

0.1

73

0.1

83

0.1

84

0.1

84

–0.1

93

0.0

01

0.2

41

0.2

14

0.2

15

0.2

23

0.2

03

0.2

02

0.2

59

0.2

35

0.2

51

0.2

39

0.2

48

0.2

55

0.2

24

0.2

63

0.2

32

0.2

69

0.2

41

0.2

53

0.2

26

0.2

29

0.2

54

0.2

65

0.2

39

7.

Lim

nia

ott

aw

ensi

s0.1

68

0.1

59

0.1

80

0.1

56

0.1

73

0.1

65

–0.1

91

0.2

22

0.2

12

0.1

79

0.1

99

0.1

96

0.1

42

0.2

49

0.2

26

0.2

54

0.2

27

0.2

21

0.2

25

0.1

85

0.2

56

0.1

90

0.2

11

0.2

20

0.2

67

0.1

79

0.1

72

0.2

11

0.2

33

0.1

93

8.

Lim

nia

sand

ova

lensi

s0.1

83

0.1

76

0.1

87

0.1

84

0.1

85

0.0

05

0.1

68

–0.2

39

0.2

12

0.2

17

0.2

21

0.2

02

0.2

03

0.2

61

0.2

36

0.2

53

0.2

41

0.2

47

0.2

53

0.2

23

0.2

62

0.2

30

0.2

68

0.2

40

0.2

52

0.2

24

0.2

27

0.2

56

0.2

63

0.2

38

9.

Renocera

am

and

a0.2

12

0.2

01

0.2

10

0.2

10

0.2

09

0.2

24

0.2

00

0.2

23

–0.2

38

0.2

29

0.2

20

0.2

10

0.2

45

0.2

70

0.2

36

0.2

48

0.2

35

0.2

54

0.2

34

0.2

08

0.2

56

0.2

15

0.2

28

0.2

44

0.2

66

0.2

42

0.2

26

0.2

51

0.2

47

0.2

47

10.

Renocera

johnso

ni

0.2

04

0.1

91

0.1

97

0.1

84

0.2

05

0.1

90

0.1

84

0.1

90

0.2

27

–0.2

20

0.2

32

0.2

26

0.2

27

0.2

61

0.2

32

0.2

42

0.2

38

0.2

44

0.2

42

0.2

42

0.2

72

0.2

24

0.2

54

0.2

45

0.2

65

0.2

24

0.2

11

0.2

32

0.2

54

0.2

30

11.

Sep

ed

on

arm

ipes

0.1

77

0.1

77

0.1

86

0.1

79

0.1

99

0.1

89

0.1

56

0.1

94

0.2

12

0.1

95

–0.1

82

0.1

67

0.2

03

0.2

59

0.2

23

0.2

72

0.2

27

0.2

21

0.2

37

0.2

30

0.2

36

0.2

00

0.2

28

0.2

08

0.2

50

0.2

02

0.2

00

0.2

08

0.2

45

0.2

21

12.

Sep

ed

on

fusc

ipennis

0.1

84

0.1

61

0.1

87

0.1

73

0.1

98

0.1

95

0.1

61

0.1

94

0.2

12

0.1

94

0.1

51

–0.1

84

0.2

05

0.2

53

0.2

30

0.2

60

0.2

35

0.2

36

0.2

26

0.2

16

0.2

47

0.1

93

0.2

11

0.2

41

0.2

34

0.2

03

0.1

85

0.2

35

0.2

62

0.2

15

13.

Sep

ed

on

pra

em

iosa

0.1

88

0.1

84

0.1

90

0.1

79

0.2

00

0.1

93

0.1

61

0.1

95

0.2

15

0.2

00

0.1

47

0.1

60

–0.2

33

0.2

56

0.2

32

0.2

54

0.2

35

0.2

30

0.2

29

0.2

24

0.2

65

0.1

88

0.2

32

0.2

40

0.2

64

0.2

09

0.2

03

0.2

21

0.2

56

0.2

17

14.

Try

peto

pte

racanad

ensi

s0.1

98

0.1

86

0.1

91

0.1

79

0.1

87

0.1

80

0.1

51

0.1

80

0.2

25

0.2

02

0.2

04

0.1

98

0.1

98

–0.2

44

0.2

21

0.2

69

0.2

21

0.2

50

0.2

44

0.1

93

0.2

72

0.2

11

0.2

37

0.2

38

0.2

46

0.2

08

0.1

97

0.2

14

0.2

51

0.2

06

15.

Try

peto

pte

raarr

ogans

0.2

20

0.2

08

0.2

12

0.2

14

0.2

17

0.2

24

0.2

05

0.2

21

0.2

36

0.2

27

0.2

27

0.2

11

0.2

25

0.2

16

–0.2

38

0.2

62

0.2

40

0.2

80

0.2

61

0.2

52

0.2

71

0.2

49

0.2

63

0.2

64

0.2

48

0.2

45

0.2

53

0.2

50

0.2

82

0.2

74

16.

Try

peto

pte

rab

erg

i0.1

99

0.1

95

0.1

88

0.1

78

0.1

91

0.1

99

0.1

92

0.1

98

0.2

17

0.1

88

0.1

99

0.1

70

0.1

94

0.2

08

0.2

07

–0.2

45

0.0

10

0.2

08

0.2

10

0.1

94

0.2

60

0.2

14

0.2

35

0.2

20

0.2

11

0.1

82

0.2

15

0.1

93

0.2

36

0.2

08

17.

Try

peto

pte

racla

ra0.2

34

0.2

13

0.2

13

0.2

03

0.2

07

0.2

21

0.2

11

0.2

21

0.2

31

0.1

89

0.2

23

0.2

04

0.2

28

0.2

21

0.2

05

0.2

10

–0.2

41

0.2

45

0.2

40

0.2

45

0.2

57

0.2

23

0.2

49

0.2

45

0.2

53

0.2

44

0.2

50

0.2

51

0.2

21

0.2

47

18.

Try

peto

pte

rafe

rrugin

ea

0.2

01

0.1

92

0.1

89

0.1

79

0.1

94

0.1

97

0.1

92

0.1

96

0.2

20

0.1

89

0.2

00

0.1

73

0.1

96

0.2

11

0.2

10

0.0

11

0.2

10

–0.2

03

0.2

13

0.1

97

0.2

60

0.2

20

0.2

36

0.2

25

0.2

05

0.1

90

0.2

18

0.2

02

0.2

42

0.2

05

19.

Teta

nocera

fusc

inerv

is0.2

04

0.1

93

0.1

93

0.1

88

0.1

96

0.1

85

0.1

83

0.1

85

0.2

28

0.2

10

0.2

00

0.1

88

0.2

01

0.2

04

0.2

03

0.1

77

0.2

05

0.1

77

–0.2

25

0.2

12

0.2

26

0.2

29

0.2

26

0.2

04

0.2

51

0.1

93

0.2

06

0.2

32

0.2

50

0.1

96

20.

Teta

nocera

kert

esz

i0.2

18

0.2

13

0.1

93

0.1

93

0.1

94

0.2

10

0.2

00

0.2

13

0.2

17

0.2

03

0.2

11

0.1

98

0.2

18

0.2

19

0.2

18

0.1

97

0.1

91

0.1

97

0.1

87

–0.1

98

0.2

45

0.1

99

0.1

36

0.2

25

0.2

46

0.1

99

0.2

08

0.2

23

0.2

44

0.2

23

21.

Teta

nocera

latifi

bula

0.2

04

0.2

04

0.1

86

0.1

85

0.1

83

0.1

92

0.1

75

0.1

91

0.2

06

0.1

94

0.1

93

0.1

93

0.2

03

0.2

04

0.2

06

0.1

68

0.1

89

0.1

72

0.1

87

0.1

68

–0.2

35

0.1

61

0.1

96

0.2

11

0.2

38

0.1

66

0.1

87

0.1

96

0.2

38

0.1

91

22.

Teta

nocera

mela

nost

igm

a0.2

58

0.2

43

0.2

34

0.2

30

0.2

31

0.2

34

0.2

40

0.2

38

0.2

62

0.2

24

0.2

58

0.2

43

0.2

53

0.2

42

0.2

13

0.2

29

0.2

17

0.2

29

0.2

26

0.2

24

0.2

26

–0.2

39

0.2

50

0.2

53

0.2

75

0.2

59

0.2

32

0.2

62

0.2

36

0.2

53

23.

Teta

nocera

meso

pora

0.1

90

0.1

97

0.1

88

0.1

81

0.1

87

0.1

98

0.1

80

0.1

97

0.2

07

0.1

89

0.1

94

0.1

86

0.1

97

0.2

04

0.2

16

0.1

81

0.1

99

0.1

79

0.1

86

0.1

76

0.1

62

0.2

34

–0.2

01

0.2

02

0.2

46

0.1

66

0.2

03

0.1

88

0.2

32

0.1

78

24.

Teta

nocera

monta

na

0.2

14

0.2

11

0.1

85

0.1

93

0.1

94

0.2

11

0.1

91

0.2

12

0.2

19

0.2

01

0.2

08

0.1

96

0.2

15

0.2

12

0.2

10

0.1

81

0.1

88

0.1

79

0.1

90

0.1

02

0.1

68

0.2

27

0.1

67

–0.2

17

0.2

69

0.1

90

0.2

11

0.2

10

0.2

56

0.2

22

25.

Teta

nocera

phyl

lop

hora

0.2

12

0.2

04

0.1

92

0.1

88

0.2

05

0.1

95

0.1

82

0.1

99

0.2

23

0.1

90

0.1

90

0.1

86

0.2

04

0.2

03

0.2

16

0.1

81

0.1

96

0.1

81

0.1

90

0.1

91

0.1

74

0.2

29

0.1

70

0.1

92

–0.2

50

0.1

98

0.2

14

0.1

95

0.2

39

0.2

13

26.

Teta

nocera

ple

beja

0.2

19

0.2

02

0.1

99

0.2

16

0.1

98

0.1

88

0.1

99

0.1

92

0.2

38

0.2

17

0.2

17

0.2

14

0.2

21

0.2

16

0.2

00

0.1

88

0.2

10

0.1

88

0.1

92

0.2

06

0.1

99

0.2

28

0.2

04

0.2

06

0.1

95

–0.2

41

0.2

49

0.2

34

0.2

69

0.2

56

27.

Teta

nocera

plu

mosa

0.1

97

0.1

86

0.1

82

0.1

82

0.1

91

0.1

88

0.1

81

0.1

90

0.2

13

0.1

92

0.1

91

0.1

88

0.1

93

0.2

04

0.2

16

0.1

70

0.2

01

0.1

71

0.1

92

0.1

86

0.1

63

0.2

27

0.1

62

0.1

74

0.1

82

0.1

99

–0.1

70

0.1

78

0.2

45

0.1

70

28.

Teta

nocera

rob

ust

a0.1

74

0.1

66

0.1

65

0.1

62

0.1

77

0.1

77

0.1

57

0.1

79

0.2

07

0.1

88

0.1

81

0.1

75

0.1

88

0.1

89

0.2

21

0.1

81

0.1

91

0.1

80

0.1

74

0.1

81

0.1

68

0.2

26

0.1

67

0.1

79

0.1

72

0.1

99

0.1

72

–0.2

08

0.2

21

0.1

66

29.

Teta

nocera

silv

atic

a0.1

85

0.1

72

0.1

70

0.1

70

0.1

79

0.1

88

0.1

61

0.1

90

0.2

13

0.1

86

0.1

86

0.1

90

0.1

84

0.1

87

0.2

03

0.1

80

0.2

08

0.1

79

0.1

79

0.1

79

0.1

85

0.2

21

0.1

75

0.1

84

0.1

84

0.2

01

0.1

79

0.1

59

–0.2

27

0.1

99

30.

Teta

nocera

valid

a0.2

19

0.1

95

0.2

00

0.1

96

0.2

00

0.2

00

0.1

91

0.2

00

0.2

27

0.1

92

0.2

19

0.1

95

0.2

16

0.2

16

0.2

07

0.1

94

0.1

95

0.1

95

0.1

90

0.1

96

0.2

01

0.2

23

0.1

86

0.1

87

0.1

88

0.1

95

0.1

94

0.1

95

0.1

95

–0.2

39

31.

Teta

nocera

vicin

a0.1

90

0.1

77

0.1

76

0.1

81

0.1

84

0.1

91

0.1

74

0.1

94

0.2

05

0.1

90

0.1

89

0.1

84

0.1

86

0.2

02

0.2

09

0.1

77

0.2

02

0.1

77

0.1

96

0.1

90

0.1

62

0.2

20

0.1

74

0.1

77

0.1

81

0.2

09

0.1

51

0.1

76

0.1

75

0.1

86

–

Low

er

left

,C

OI

p-d

ista

nce

s;u

pper

righ

t,C

OII

p-d

ista

nce

s.

1466 E. G. CHAPMAN ET AL.

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To further test the robustness of our phylogenetic

inferences, we performed a variety of topology tests

comparing the best unconstrained tree topology with a

tree in which the terrestrial Tetanocera were constrained to

be monophyletic. The best ML constrained tree was

identical to the unconstrained ML topology, except for

the arrangement of Tetanocera species (tree not shown).

All tests strongly rejected the null hypothesis that the

difference between the two trees was no greater than

expected from sampling error (P < 0.0001 for all; Table 5).

Habitat-morphology correlations

Figure 5 shows four mirror trees visualizing the correla-

tions between larval habitat (left side of each tree) and

each of four morphological characters: (i) larval colour

(Fig. 5a), (ii) float hair length (Fig. 5b), (iii) ventrolateral

lobe length ratios (Fig. 5c), (iv) and orientation of the

posterior spiracular disc (Fig. 5d), each on the right side

of their respective trees. The parsimony-inferred ances-

tral character states for Tetanocera are (i) aquatic habitat,

(ii) dark colour, (iii) long float hairs, (iv) relatively long

ventrolateral lobes and (v) lengthened last abdominal

segment with upturned spiracular disc (Fig. 5; the

orientation of the spiracular disc in Tetanocera silvatica

could not be determined, therefore was coded as

unknown in Fig. 5d). Thus, the initial direction of change

for the polarized characters within Tetanocera was from (i)

aquatic to terrestrial habitat, (ii) dark to light colour, (iii)

long to short float hairs, (iv) from relatively long to

Fig. 3 Best tree topology (as judged by ML)

from a sample of 1001 trees from a Bayesian

analysis of the concatenated dataset, show-

ing posterior probabilities above the lines and

ML bootstrap and MP bootstrap values below

the lines, the latter in parentheses. Numbers

in parentheses after the taxon names indi-

cate how many specimens of each species

were sequenced and analysed. Genus

abbreviations from top to bottom: Sciomyz-

ini: Atrichomelina (A); Sciomyza (S); Tetan-

ocerini: Sepedon (S); Elgiva (E); Hedria (H);

Limnia (L); Trypetoptera (T); Renocera (R);

Tetanocera (T).

Habitat correlated parallel evolution 1467

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relatively short ventrolateral lobes and (v) from upturned

to rear-facing spiracular disc.

The trees in Fig. 5 illustrate the relatively tight corre-

lations we observed between habitat and morphology.

Dark colour is perfectly correlated with aquatic habitat

within Tetanocera; the outgroup genus Sepedon is the

exception (Fig. 5a). The only exception to a perfect

correlation between aquatic habitat and long float hairs

occurs if Tetanocera plumosa (Fig. 5b) is considered aqua-

tic. There is a perfect correlation between aquatic habitat

and relatively long ventrolateral lobes throughout the

entire phylogeny (Fig. 5c). Finally, the orientation of the

spiracular disc has two mismatches among the outgroup

genera (Hedria and Renocera) and a single mismatch

within Tetanocera (T. fuscinervis; Fig. 5d).

The correlated evolution tests between habitat and the

above four morphological characters all yielded signifi-

cant correlations (P £ 0.01), as did tests of correlations

among morphological characters (P £ 0.02; Table 6). All

correlations were significant, and all remained significant

after applying the sequential Bonferroni test. Further-

more, of the 18 possible temporal order tests among the

four morphological characters (coding T. silvatica with or

without an upturned spiracular disc, and examining both

the entire phylogeny and the Tetanocera portion), only

one was significant: cuticular pigmentation is reduced

before float hair length is shortened on the entire

phylogeny (v21 ¼ 4.76, P ¼ 0.029). Of the 20 possible

temporal order tests between habitat and the four

morphological characters (coding T. silvatica with or

without an upturned spiracular disc, coding T. plumosa

aquatic or terrestrial, and examining both the entire

phylogeny and the Tetanocera portion), only one was

significant: habitat changes from aquatic to terrestrial

before cuticular pigmentation is reduced on the entire

phylogeny if T. plumosa is coded as terrestrial (v21 ¼ 5.17,

P ¼ 0.023). Neither of these results remained significant

after applying the sequential Bonferroni test.

Discussion

The results of our phylogenetic and correlated evolution

analyses indicate that larval habitat changes are robustly

linked to larval pigmentation, float hair length, spiracular

disc orientation, and ventrolateral lobe length. These

analyses are consistent with the hypothesis that terrest-

rial habitat preference and the morphological character

states typical of terrestrial Tetanocera species all arose at

least three times independently (Figs 3 and 4). Knutson

& Vala (2002) conclude ‘terrestrial behaviour and mor-

phology in the Tetanocerini are apomorphic features of

that tribe’. Our species-level phylogeny, ancestral char-

acter state optimizations, and topology tests clearly

support this conclusion with respect to Tetanocera.

Habitat–morphology correlations

Because our optimization analyses (Figs 3 and 4) indicate

at least three independent transitions from aquatic to

terrestrial larval habitat within Tetanocera, and all corre-

lated evolution tests were significant, we are able to infer

that changes in habitat preference were typically accom-

panied by morphological transitions in four larval char-

acters. All four of these morphological character state

changes can be viewed as transitions to more vestigial

conditions, as they are either a reduction of cuticular

pigmentation or a reduction in the size of the structure. In

his comprehensive work on the breeding habits and

immature stages of cyclorrhaphan Diptera (the division to

which the Sciomyzidae belongs), Ferrar (1987) stated that

Fig. 4 Maximum likelihood optimizations of

habitat analysed with MESQUITEMESQUITE: (a) asym-

metrical two-parameter model, (b) MK1

model. An asterisk (*) to the left of a node

indicates significance (ancestral states with

raw likelihood scores >2.0).

1468 E. G. CHAPMAN ET AL.

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Fig. 5 Mirror trees showing the correlations between habitat and (a) larval colour, (b) float hair length, (c) ventrolateral lobe ratios and (d)

orientation of the posterior spiracular disc. Character optimizations done using equally weighted parsimony methods in MESQUITEMESQUITE.

Table 5 Results of the parsimony-based Kishino-Hasegawa (KH), Templeton (Wilcoxon signed-ranks) and winning sites (sign) tests calculated

using PAUPPAUP*, and the likelihood-based approximately unbiased (AU), Kishino–Hasegawa (KH), Shimodiara-Hasegawa (SH), weighted Kishino-

Hasegawa (WKH), and weighted Shimodiara-Hasegawa (WSH) tests calculated using CONSEL. Trees compared were the best topology from the

unconstrained Bayesian analysis vs. an analysis where the terrestrial Tetanocera were constrained to be monophyletic.

Tree

Test

Length Difference KH Templeton Winning sites

Parsimony-based tests

Unconstrained 8506

Terrestrials constrained 8719 213 P < 0.0001 P < 0.0001 P < 0.0001

)Ln L Difference AU KH SH WKH WSH

Likelihood-based tests

Unconstrained )37275.65

Terrestrials constrained )37587.04 311.39 P ¼ 3e)22 P ¼ 0 P ¼ 0 P ¼ 0 P ¼ 0

Habitat correlated parallel evolution 1469

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‘I still believe that to some extent one can draw conclu-

sions on [phylogenetic] relationship from immature

stages, but I now consider that larval morphology is

predominantly functional and that larvae show a number

of interesting examples of parallel evolution’. Our analy-

ses of Tetanocera phylogenesis and character evolution,

which quite strikingly corroborate Ferrar’s conclusion,

indicate at least three instances where larval morphology

changed in the same way during habitat shifts.

Table 7 lists the character states of the Tetanocera

species with known habitat preferences that we were

unable to include in the analyses presented herein. Upon

examination of these data, it is clear that no matter

where these species eventually insert into the phylogeny

of Tetanocera, they will only render our highly significant

habitat–morphology correlations stronger. Furthermore,

given the high nodal support values for the three

Tetanocera subclades and the highly significant results of

the constrained/unconstrained topology tests, it seems

very unlikely that the addition of these taxa would

rearrange the phylogeny such that the aquatic and/or

terrestrial species would form monophyletic groups.

Thus, the parallel evolution of terrestrial habit and

associated morphological changes appears to be the best

explanation for the observed character distributions

within Tetanocera.

Because of the tight correlations among the morpho-

logical characters examined (i.e. all temporal order tests

were nonsignificant after applying the sequential Bon-

ferroni test), we were not able to distinguish among three

possible scenarios regarding these correlations: (i) plei-

otropy, (ii) indirect selection via another trait, or (iii)

concerted evolution. However, the two habitat–mor-

phology mismatches within Tetanocera may be indicative

of the general ordering of larval morphological character

state transitions or of current adaptation to novel

habitats. Tetanocera plumosa has larvae that are principally

aquatic but can also be found in shoreline (¼terrestrial)

situations (Foote, 1961). This species exhibits one mor-

phological character state (short float hairs) that is

typically found in terrestrial larvae. Tetanocera fuscinervis,

a shoreline predator, has an upturned spiracular disc,

Table 6 Tests for correlated evolution using DISCRETEDISCRETE (omnibus test) showing likelihood ratios (LR) and associated P-values for each test.

Habitat–morphology correlations Habitat vs.

Scenario Pigmentation Float hair length Ventrolateral lobe length Spiracular disc orientation

Tetanocera plumosa coded aquatic

Entire phylogeny LR ¼ 10.40, P < 10)6 LR ¼ 13.77, P < 10)6 LR ¼ 13.44, P < 10)6 LR ¼ 10.11, P < 10)6

Tetanocera portion only LR ¼ 10.59, P < 10)6 LR ¼ 8.09, P < 10)6 LR ¼ 10.59, P < 10)6 LR ¼ 8.06, P < 10)6

T. plumosa coded terrestrial

Entire phylogeny LR ¼ 6.86, P < 10)6 LR ¼ 16.21, P < 10)6 LR ¼ 10.42, P < 10)6 LR ¼ 8.30, P < 10)6

Tetanocera portion only LR ¼ 8.02, P < 10)6 LR ¼ 9.86, P < 10)6 LR ¼ 8.11, P < 10)6 LR ¼ 6.57, P < 10)6

T. silvatica with upturned disc

Entire phylogeny N/A N/A N/A LR ¼ 6.70, P £ 0.01

Tetanocera portion only N/A N/A N/A LR ¼ 5.34, P £ 0.01

T. silvatica with rear-facing disc

Entire phylogeny N/A N/A N/A LR ¼ 8.30, P < 10)6

Tetanocera portion only N/A N/A N/A LR ¼ 6.37, P < 10)6

Morphology–morphology correlations

Scenario Pigmentation Float hair length Ventrolateral lobe length

Float hair length

Entire phylogeny LR ¼ 10.46, P < 10)6 – –

Tetanocera portion only LR ¼ 8.09, P < 10)6 – –

Ventrolateral lobe length

Entire phylogeny LR ¼ 11.62, P < 10)6 LR ¼ 10.42, P < 10)6 –

Tetanocera portion only LR ¼ 10.43, P < 10)6 LR ¼ 8.11, P < 10)6 –

Spiracular disc orientation

T. silvatica with upturned disc

Entire phylogeny LR ¼ 6.03, P < 10)6 LR ¼ 6.70, P < 10)6 LR ¼ 8.38, P < 10)6

Tetanocera portion only LR ¼ 6.37, P < 10)6 LR ¼ 5.33, P £ 0.02 LR ¼ 6.38, P £ 0.01

T. silvatica with rear-facing disc

Entire phylogeny LR ¼ 8.33, P < 10)6 LR ¼ 8.30, P < 10)6 LR ¼ 8.38, P < 10)6

Tetanocera portion only LR ¼ 8.08, P < 10)6 LR ¼ 6.57, P < 10)6 LR ¼ 8.07, P < 10)6

Top half, correlation tests between habitat (aquatic or terrestrial) and four morphological characters under a number of different possible scenarios.

Correlated evolution tests were conducted on both the entire phylogeny and the Tetanocera portion only. Because T. plumosa can be found in both

aquatic and shoreline habitats, both scenarios were tested. Because the spiracular disc orientation of T. silvatica could not be determined, both

character states were tested. Each test was repeated to insure accuracy; Bottom half, correlation tests between morphological characters.

1470 E. G. CHAPMAN ET AL.

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which is the typical character state in aquatic species.

These two exceptions to the rule could represent the two

ends of an aquatic-to-terrestrial transition series. For

example, the reduction of float hair length and the

evolution of a posterior-facing spiracular disc could be

the first and last, respectively, of the four characters to

change during the evolutionary transition to a terrestrial

larval habit in Tetanocera. This interpretation suggests that

the larval stage of T. plumosa may be in the initial phase of

transitioning to land and thus the short float hairs

represent an adaptation to the much drier shoreline

habitat. Furthermore, T. fuscinervis larvae could be

viewed as being in the penultimate phase of transitioning

to land with the upturned spiracular disc being viewed as

historical baggage. The observation that the two, possibly

transitional, Tetanocera species possessing mismatched

character states occur in shoreline situations is consistent

with a stepping stone role for this habitat in aquatic to

terrestrial habitat transitions. Alternatively, these hab-

itat–morphology mismatches may represent currently

advantageous characteristics for specialized ecological

circumstances. This hypothesis seems reasonable due to

the ecotonal nature of the shoreline habit in these two

species. For example, the upturned spiracular disc in

T. fuscinervis could allow this species to breathe during

periodic flooding of its shoreline habitat, enabling it to

swim back to the shoreline. Ongoing, taxonomically

more inclusive, phylogenetic/PCM studies within the

Sciomyzidae, combined with evaluations of current

utility for the mismatched character states within Tetano-

cera, offer possible means to evaluate these hypotheses.

Both ancestral character state optimization methods

suggested one reversal from terrestrial to aquatic habit in

Tetanocera (Figs 4 and 5). Three parallel shifts to terrest-

rial habit plus one reversal is a more parsimonious

explanation than the five parallel shifts to terrestrial habit

that would be required if the ancestor to the phyllophora-

valida clade were aquatic. Considering that the four

morphological character states of the two species in

question (T. ferruginea and T. bergi) closely resemble those

of other aquatic species, this habitat reversal suggests that

this lineage, minimally, reacquired cuticular pigmenta-

tion, long float hairs and long ventrolateral lobes. As a

lengthened last abdominal segment with upturned spir-

acular disc occurs in the extant shoreline-inhabiting

T. fuscinervis (the sister taxon to T. bergi + T. ferruginea), it

may be that the T. ferruginea + T. bergi lineage moved to

the shoreline and then back into the water without ever

changing this character. Whiting et al. (2003) demon-

strated that fully developed wings re-evolved in as many

as four independent lineages of stick insects. His study

demonstrated that a complex structure such as wings,

requiring interactions between nerves, muscles, sclerites,

and wing blades, could be lost and subsequently reac-

quired. In this light, the reacquisition of aquatic habitat-

associated morphologies in Tetanocera is not surprising,

especially if the genes responsible for the formation of

these traits are not functionally ‘degraded’ during and

after previous habitat transitions.

Phylogenetic niche conservatism

Pigmented cuticle, long float hairs, long ventrolateral

lobes, and a lengthened last abdominal segment with an

upturned spiracular disc are the plesiomorphic character

states for Tetanocera (Fig. 5a–d). There are two possible

explanations for the retention of plesiomorphic traits

within a lineage: (i) phylogenetic constraint (McKitrick,

1993; Brooks & McLennan, 1994) or (ii) phylogenetic

niche conservatism (Harvey & Pagel, 1991). McKitrick

(1993) defined phylogenetic constraint as ‘any result or

component of the phylogenetic history of a lineage that

prevents an anticipated course of evolution in that

lineage’. Phylogenetic constraint may result from the

lack of genetic variation for a given trait, from coadap-

tation among traits that impose a genetic burden on the

trait of interest, or from developmental correlations

among traits (Wagner, 1995; Givnish, 1997). These

constraints often result in a lag between environmental

shifts and phenotypic change (Johnson et al., 1999). On

the other hand, phylogenetic niche conservatism

involves the action of stabilizing selection on phenotypic

traits when ancestral ecological conditions are main-

tained within a lineage (Lord et al., 1995). Given that (i)

there was a minimum of three independent shifts to

terrestrial habit within Tetanocera, each accompanied by

Table 7 Character states of the Tetanocera species with known

habitat preference not included in the phylogeny presented herein.

Length Pigmentation

Float hair

length

Ventrolateral

lobe length

Spiracular

disc

orientation

Aquatic species

T. annae 0 0 0 0

T. loewi 0 0 0 0

T. obtusifibula 0 0 0 0

T. punctifrons 1 0 0 0

T. soror 0 0 0 0

T. spreta 0 0 0 0

T. stricklandi 0 0 0 0

Terrestrial species

T. elata 1 1 1 1

T. hyalipennis 1 1 1 ?

T. oxia 1 1 1 1

T. rotundicornis 1 1 1 1

T. spirifera 1 1 1 1

State 0, darkly pigmented, long float hairs, long ventrolateral lobes,

and lengthened terminal abdominal segment with upturned disc;

State 1, unpigmented (appearing white), very short float hairs, short

ventrolateral lobes, and terminal abdominal segment not length-

ened, disc not upturned. Data from Foote (1961); unpublished data)

and Knutson (1963).

Habitat correlated parallel evolution 1471

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the same changes in these four morphological characters

(clearly demonstrating the ability to evolve), (ii) all

temporal order tests of habitat and morphology were

nonsignificant and (iii) each plesiomorphic character

state is apparently fundamental to aquatic existence, it

seems apparent that phylogenetic niche conservatism,

rather than phylogenetic constraint, continues to main-

tain these ancestral morphologies in the extant aquatic

Tetanocera species.

Adaptation to terrestrial existence

Multiple parallel morphological character state changes

accompanying a given habitat shift can be considered as

evidence that the characters are adaptive (Baum &

Larson, 1991; Brooks & McLennan, 1991,1994; Givnish,

1997; Patterson & Givnish, 2002). We are interpreting

these multiple parallel changes in Tetanocera larval

morphology, which are significantly correlated with

habitat, as evidence consistent with an adaptation hypo-

thesis. Extrapolation from a correlative to a cause-and-

effect relationship is difficult in historical evolutionary

studies as random associations of character states can

potentially account for any observed pattern. However,

we hypothesize that the inferred changes in the four

morphological characters that accompanied the three

transitions from the aquatic to the terrestrial habitat

represent adaptive peak shifts, as each of the aquatic

character states may typically be selected against in the

terrestrial setting (e.g. Harvey & Pagel, 1991, Martins,

2000). For instance, the long ventrolateral lobes and

upturned posterior spiracular discs of aquatic Tetanocera

larvae may generally be impediments to prey-seeking

movements in the vegetative tangles of the terrestrial

habitat. An alternative cause-and-effect relationship, in

which changes in larval morphology caused habit

transitions, seems highly unlikely due to the physiologi-

cal requirements of aquatic sciomyzid larvae.

Studies of Anolis lizards in the Greater Antilles demon-

strated that distinct lineages evolved similar morphologies

as they made similar habitat shifts on different islands

(Losos, 1992; Losos et al., 1994,1998). At least 17 parallel

habitat transitions resulted in similar communities on

each island. This landmark work demonstrated that

similar solutions to similar evolutionary problems can

be arrived at multiple times independently. Tetanocera

exhibits a comparable phenomenon in that multiple

lineages have transitioned to terrestrial larval foraging

during phylogenesis (Figs 3 and 4) with each transition

typically accompanied by the same four changes in larval

morphology. These derived character states likely repre-

sent adaptations to the terrestrial environment.

One of the interesting evolutionary questions gener-

ated by this study is ‘why did the larvae of multiple

Tetanocera lineages leave the water for a terrestrial

existence?’ This is not a general evolutionary tendency

for the tribe as several tetanocerine genera have

exclusively aquatic larvae (e.g. Dictya, Elgiva, and Sepedo-

nea). There are biotic and abiotic factors that may have

been involved in these habitat transitions. Potential biotic

factors for the transition from the aquatic to the terrest-

rial larval habit in Tetanocera include the following: (i)

eliminating competition with other aquatic pulmonate

snail predators, (ii) escaping aquatic predators/parasi-

toids, (iii) compensating for prolonged declines in aquatic

pulmonate snail populations and (iv) low dispersal

ability. Very little is known about the current levels of

competition, predation, and dispersal ability, let alone

about the historical levels, that act or have acted upon

the larval stages of Tetanocera.

An abiotic factor that could influence transitions from

aquatic to terrestrial habitats is a generally drying

climate. This could markedly affect the fitness of aquatic

Tetanocera larvae directly by reducing the amount of

suitable aquatic habitat. Wiegmann et al. (2003) estima-

ted that the Schizophora [the dipteran subclade (series)

to which the Sciomyzidae belongs] arose between

142 and 70 mya with the oldest known fossil sciomyzid

dated to the Eocene/Oligocene epochs (55-24 mya;

Hennig, 1965) and the oldest known fossil Tetanocera

from the Oligocene epoch (34-24 mya; Theobald, 1937;

Forster, 1891). The time period between the beginning of

the Cenozoic era through the end of the Pliocene epoch

(65-5 mya) is generally characterized by an increasingly

cooler and drier climate. As wetlands began to dry (or at

least change from permanent to temporary wetlands),

sciomyzid lineages were likely pressured to cope with

such conditions, and some populations may have

responded by gradually shifting to more and more

terrestrial habitats. The multiple aquatic-to-terrestrial

larval habitat transitions identified herein for Tetanocera

may have occurred during this time period in response to

this general climatic trend. Additionally, because not all

tetanocerine genera display aquatic to terrestrial habitat

transitions (e.g. Dictya), factors intrinsic to Tetanocera may

also be potentiating the changes (e.g. dispersal ability).

However, lineages such as Dictya may have diversified

after the drying time period, but the lack of fossils for

such lineages prevents us from speculating further.

Subsequent phylogenetic/comparative investigations,

including those concentrating on dating the behavioural,

ecological, and morphological changes we have des-

cribed, will likely be of significant utility in further

elucidating the causal processes responsible for these

evolutionary transitions.

Acknowledgments

We thank Lita Greve-Jensen, Lloyd Knutson, Wayne

Mathis, Rory McDonnell, Joe Keiper, Ladislav Roller, and

Rudolph Rozkosny for donating specimens for this

project. We thank Diana Senyo, Judy Santmire, and

Jennifer Walker for helping with lab work. We thank

Bob Androw, Steve Chordas III, Laura Lawler-Chapman,

1472 E. G. CHAPMAN ET AL.

ª 2 0 0 6 T H E A U T H O R S 1 9 ( 2 0 0 6 ) 1 4 5 9 – 1 4 7 4

J O U R N A L C O M P I L A T I O N ª 2 0 0 6 E U R O P E A N S O C I E T Y F O R E V O L U T I O N A R Y B I O L O G Y

Joe Keiper, Austin Richards and Rob Roughley for

assistance in the field. This work was partially funded

by NSF grant DEB-0237175 (to W.R.H.), and by a Kent

State University Graduate Student Senate research grant.

We thank Ferenc DeSzalay, Joe Keiper, Patrick Lorch,

Austin Richards, Andrea Schwarzbach, and Jennifer

Walker for reviewing drafts of this manuscript. Finally,

we thank Allen Moore and the anonymous reviewer for

their comments on the manuscript.

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2006

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