Diversity of features of the female reproductive system and othermorphological characters in leeches (Citellata, Hirudinida) in

phylogenetic conception

Aleksander Bieleckia,*, Piotr �Swiaztekb, Joanna M. Cichockaa, Mark E. Siddallc,Anna Z. Urbiszb and Bartosz J. Płachnod

aDepartment of Zoology, University of Warmia and Mazury, Oczapowskiego 5, 10-719, Olsztyn, Poland; bDepartment of Animal Histology and

Embryology, University of Silesia, Bankowa 9, 40-007, Katowice, Poland; cDivision of Invertebrate Zoology, American Museum of Natural History,

New York, NY, USA; dDepartment of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31- 044, Cracow, Poland

Accepted 30 August 2013

Abstract

An epistemological–evolutionary conception of leeches (Hirudinida) based on features of the female reproductive system incombination with other morphological characters is presented in the spirit of the cladistic school of taxonomy. Characters relat-ing to the structure of the ovary and the course of oogenesis in leeches were interpreted in this manner, for the first time. Eachstudy was conducted on type species of higher taxonomic groups of true leeches. Results of analyses using features of the repro-ductive system only as well as in combination with other morphological characters show Piscicolidae and Glossiphoniidae as sis-ter clades making Rhynchobdellida a monophyletic group. Also, Hirudiniformes and Erpobdelliformes appeared to be sisterclades within Arhynchobdellida. The relationship between the outgroup specimens and leeches remained unresolved, becauseboth Acanthobdella peledina and branchiobdellidans appeared to be in an equivocal relationship to hirudinidans. Characters con-cerning the structure of the female reproductive system and course of oogenesis thus appeared to be useful, although conserva-tive, for reconstruction of leech phylogeny, and they well reflect phylogenetic relationships of Hirudinida at the family level.© The Willi Hennig Society 2013.

The taxonomy of leeches (Hirudinida) has been influ-enced strongly by various research traditions (increas-ingly in the area of phylogenetic reconstruction) andyet still presents numerous problems to researchers(Sereno, 2007; Cichocka and Bielecki, 2008). Nonethe-less, and in terms of the theory of biological systematicsand phylogeny, it is no longer deemed necessary to passfrom descriptions of species through to classification tosuggest archetypes and a history of their transforma-tion (Bielecki and Epstein, 1994).That Clitellata is a monophyletic group of annelids

is strongly supported by morphological and DNAsequence data (Purschke et al., 1993; Nielsen, 1995;McHugh, 1997; Siddall et al., 2001; Ers�eus and

K€allersj€o, 2004). Although many recent papers havebeen devoted to relationships among clitellates, partic-ularly in terms of molecular data (e.g. Martin, 2001;Siddall et al., 2001, 2006; Ers�eus and K€allersj€o, 2004;Ers�eus, 2005; Jamieson, 2006; Rousset et al., 2007,2008; Marotta et al., 2008), data regarding the organ-ismal evolution of Clitellata taxa are lacking.Recent studies based on molecular data (especially

18S rRNA and COI gene sequences) suggest thatbranchiobdellidans, acanthobdellids and hirudinidsform a monophyletic group within Oligochaeta (Martin,2001; Siddall et al., 2001; Ers�eus and K€allersj€o, 2004;Marotta et al., 2008). Many studies, both morphologi-cal and molecular, have also shown that severaltraditional groups of “true” leeches (Euhirudinea=Hiru-dinida) are artificial (Siddall et al., 2006). Notable stud-ies performed by Siddall and Burreson (1998), by

*Corresponding author:E-mail address: alekb@uwm.edu.pl

CladisticsCladistics 30 (2014) 540–554

10.1111/cla.12058

© The Willi Hennig Society 2013

Apakupakul et al. (1999) and by Trontelj et al. (1999)suggest that rhynchobdellids are paraphyletic, while notalways agreeing on the specifics of that paraphyly. Eventhe well-known and widely used medicinal leeches(Hirudinidae) are not monophyletic. Sket and Trontelj(2008) suggested “…that leech taxonomy—at the specieslevel and higher—is in a revolutionary phase rightnow”. This implies a need for new morphological andmolecular data if we are to fully understand the path ofleech evolution.Most morphological studies devoted to the phylog-

eny of Hirudinida, and more widely of Clitellata, arebased on data from important sets of characters con-nected with the morphology of reproductive systemsand a few details of gametogenesis (Brinkhurst, 1982,1984a,b; Brinkhurst and Gelder, 1989; Purschke et al.,1993; Siddall and Burreson, 1995; Purschke, 2002;Marotta et al., 2003, 2008). Spermatogenesis andsperm ultrastructure appear to be particularly usefulfor phylogenetic assessment (e.g. Ers�eus and Ferraguti,1995; Ferraguti and Ers�eus, 1999; Ferraguti et al.,1999; Marotta et al., 2003, 2008; Cardini and Ferrag-uti, 2004). While it is believed that spermatozoa carryimportant phylogenetic information, even smallchanges in reproductive biology altering sperm mor-phology may lead to homoplasy (Ferraguti andErs�eus, 1999). In contrast, there have been doubts thatovarian morphology, and the course of oogenesis, isequally useful for phylogenetic analyses due to per-ceived strong correlation between those characters andlife-history strategies compounded by a paucity of data(Eckelbarger, 2006).Several studies devoted to ovarian morphology and

the course of oogenesis in true leeches have been pub-lished recently (�Swiaztek, 2005a,b, 2006, 2008; Spałek-Wołczynska et al., 2008; �Swiaztek et al., 2009, 2010a,b;Ben Ahmed et al., 2010, 2013), providing substantialnew comparative data about the internal ovarian orga-nization in leeches. Specifically, these new data includethe non-polarized ovary cords in Glossiphoniidae, eggfollicles in Piscicolidae, polarized ovary cords in Hiru-dinidae and Haemopidae and in Erpobdellidae, as wellas detailed descriptions of oogenesis (i.e. extrachromo-somal DNA bodies, multiple nucleoli and accessorynuclei were described in representatives of Glossipho-niidae).Here, we investigate whether the female reproductive

morphology, ovarian organization and details ofoogenesis provide new elements useful for leech phy-logeny analysis. The aim of this study was to test thefollowing hypothesis: characters concerning the femalereproductive system and course of oogenesis are prob-ably useful for phylogenetic reconstruction of Hirudin-ida. These new data have not previously been used inphylogenetic reconstruction. New data compiled withdetails obtained from older literature concerning the

structure of the female reproductive system of leecheswere subject to cladistics analysis. In our study weused 22 characters associated with the female repro-ductive system, ovary organization and oogenesis(interpreted on the ultrastructural, structural, topologi-cal, and behavioural levels) as well as 27 other mor-phological characters from 12 representatives of eightgenera belonging to five families of Hirudinida, as wellas Acanthobdella peledina, Branchiobdella parasita, andB. pentodonta as an outgroup.All characters were polarized according to criteria

suggested by Sereno (2007), so as to not confuse neo-morphic and transformative characters. Numerouscharacter statements that had been used by otherauthors (e.g. Apakupakul et al., 1999; Utevsky andTrontelj, 2004; Williams and Burreson, 2006) wereevaluated.

Material and methods

Material examined

Leeches used for microscopic analyses were as fol-lows.Specimens of Alboglossiphonia heteroclita (Linnaeus,

1758), Glossiphonia complanata (Linnaeus, 1758),Helobdella stagnalis (Linnaeus, 1758), Haemopis san-guisuga (Linnaeus, 1758), and Erpobdella octoculata(Linnaeus, 1758) were collected from small lakes andstreams in southern Poland.Specimens of Piscicola geometra (Linnaeus, 1758),

Piscicola margaritae Bielecki, 1997 and Piscicola sp.were collected from Cyprinus carpio caught in smalllakes near Olsztyn (northern Poland), and specimensof Cystobranchus respirans (Troschel, 1850) were col-lected from the San River near Lesko (south-easternPoland).Specimens of Erpobdella johanssoni (Johansson,

1927) Siddall, 2002 [syn. Dina punctata (Johansson,1927)] were collected in small brooks connected tosprings in north-west Tunisia, in stagnant, often tem-poral water bodies at mid to high elevations (263–1010 m a.s.l.) in 2009–2010.Specimens of Hirudo medicinalis Linnaeus, 1758

were obtained from commercial breeding, specimens ofHirudo troctina Johnson, 1816 and Limnatis nilotica(Savigny, 1822) were collected from Lebna Dam, situ-ated on the river Lebna, in north-east Tunisia in 2009.These three leech species were then bred under labora-tory conditions in 10 000-cm3 aquaria. To induceoogenesis the leeches were fed bimonthly with freshrabbit blood in a pig gut casing.Specimens of Acanthobdella peledina Grube, 1815

were collected from salmonid fishes (Thymallus thymal-lus, Salmo trutta) caught in the Aksujoki River, the

A. Bielecki et al. / Cladistics 30 (2014) 540–554 541

Peltojoki River and the Inari Lake in Lapland in 2009,and specimens of Branchiobdella parasita Braun, 1805and Branchiobdella pentodonta Whitman, 1882 werecollected from freshwater crayfish (Astacus astacus)bred in the laboratory at the Division of Zoology,Faculty of Animal Sciences, Warsaw University of LifeSciences SGGW in 2008–2009.Specimens that are only microscopically illustrated

in the paper were collected in freshwater reservoirs inOlsztyn, Poland: Alboglossiphonia hyalina (O.F.M€uller, 1774), Glossiphonia verrucata (F. M€uller,1844), Placobdella costata (Fr. M€uller, 1846), Piscicolabrylinskae Bielecki, 2001, Erpobdella testacea (Savigny1820), E. vilnensis (Liskiewicz, 1925) (see also Bieleckiet al., 2011a); in lakes of Finland: Cystobranchus mam-millatus (Malm, 1863) found on a burbot (Lota lota);and in the River Ure, Manchester, UK: P. siddalli Bie-lecki, Cios and Cichocka 2012 (for more details seeBielecki et al., 2012).

Preservation methods, measurements and dissection

Leeches were anaesthetized in 10% ethanol, thenwashed of mucus in 50% ethanol, and placed in vialswith 75% ethanol or formol – 3% aqueous solution offormalin (Pawłowski, 1936). Measurements were takenunder an Olympus SZ-ST stereomicroscope withOlympus DP 10 camera at 10–20 9 magnification.Cytochemical and microscopic methods were used as

described elsewhere (�Swiaztek, 2005a,b, 2006, 2008;�Swiaztek et al., 2007, 2009, 2010a,b, 2012; Spałek-Wołczy�nska et al., 2008; Ben Ahmed et al., 2010,2013; Urbisz et al., 2010). All specimens were fixedwith 2.5% glutaraldehyde in 0.1 M phosphate buffer(pH 7.4), then dissected. Segments with gonads, orprepared gonads only, were fixed in 2.5% glutaralde-hyde in the same buffer at room temperature for morethan 2 days. After washing in phosphate buffer, thematerial was postfixed for 2 h in 1% OsO4 in the samebuffer, dehydrated in a graded ethanol series replacedby acetone and embedded in Epon 812 (Fullam Inc.,Latham, NY, USA). Semithin sections (0.8 lm thick)stained with methylene blue were examined under anOlympus BX60 microscope equipped with a DP12 dig-ital camera and AnaliSIS 3.2 (Soft Imaging System,M€unster, Germany) software. Ultrathin sections(80 nm) were cut on a Leica ultracut UCT ultramicro-tome, contrasted with uranyl acetate and lead citrate,and examined using a Hitachi H500 electron micro-scope at 75 kV.Moreover, dissected gonads of Piscicola geometra,

Erpobdella octoculata, and Haemopis sanguisuga werefixed in 4% formaldehyde (freshly prepared from para-formaldehyde) in phosphate-buffered saline (PBS:NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4 8 mM;KH2PO4, 1.5 mM, pH 7.4) for 30–40 min at room tem-

perature and analysed under an Olympus BX60 micro-scope equipped with Nomarski differential interferencecontrast. Dissected gonads of Alboglossiphonia heteroc-lita after fixation in 4% formaldehyde in PBS werestained with DAPI (4′,6-diamidino-2-phenylindoledihydrochloride) (1 lg/mL; Sigma, St Louis, MO,USA) in PBS for 45 min at room temperature indarkness. Then, whole-mounted preparations wereexamined under an Olympus BX60 epifluorescencemicroscope equipped with appropriate filters.Additionally, the studied branchiobdellidans were

used for scanning electron microscopy (SEM) studies.Whole specimens were fixed with 2.5% glutaraldehydein a 0.1 M phosphate buffer (pH 7.4) for several days.Later, the samples that had been dehydrated in etha-nol as well as acetone series were critical-point dried inliquid CO2, and coated with gold using an JEOL-JFC1100E sputter coater. The specimens were viewed inan Hitachi S-4700 microscope (Scanning MicroscopyLaboratory of Biological and Geological Sciences, Jag-iellonian University) at 20 kV.

Morphology

A matrix of 22 characters of ovarian structure andoogenesis was compiled from several other sources inaddition to our own published data (Table 1, charac-ters 1–22). We used only those characters that werewell characterized across all considered taxa. The char-acters were adopted from Ben Ahmed et al. (2010,2013), Brumpt (1900), Gruzova and Zaichikova (1967),Epstein (1973, 1987), Elliott and Mann (1979), Sawyer(1986), Gruzova and Parfenov (1993), Siddall andBurreson (1995), Bielecki (1997, 2004), Apakupakulet al. (1999), Siekierska (2003), �Swiaztek (2005a,b, 2006,2008), Siddall et al. (2006), Williams and Burreson(2006), �Swiaztek et al. (2007, 2010a, 2012), Spałek-Wołczy�nska et al. (2008), and Urbisz and �Swiaztek(2013). The descriptions of each character is as follows:1 Oogenesis: (0) outside ovisacs; (1) within ovisacs2 Cytophore: (0) well developed (Figs 1c,e, 2f); (1)

poorly developed (Fig. 2e)3 Ovary, internal organization: (0) ovary cords

(Figs 1e, 2b–d); (1) egg follicles (Fig. 2a) [Note: inHirudinida and Acanthobdellida ovaries are com-posed from an external envelope (ovarian sac=ovi-sac) that is filled with hemocelomic fluid in whichseveral freely floating internal structures occur. Theseinternal ovarian units may have the form of long orshort ovary cords (ovary strings) or spherical egg fol-licles (for details see: �Swiaztek, 2005a,b, 2006, 2008;Ben Ahmed et al., 2010, 2013; �Swiaztek et al., 2010a,2012).]4 Ovary, polarization of the internal ovarian units:

(0) present (Fig. 2c,d); (1) absent [Note: polarizationof the internal units means that there is a prominent

542 A. Bielecki et al. / Cladistics 30 (2014) 540–554

gradient of germ-cell development within the givenunit (for details see: �Swiaztek, 2005a,b, 2006, 2008; BenAhmed et al., 2010, 2013; �Swiaztek et al., 2010a, 2012).]

5 Eggs: (0) yolky (Fig. 3b); (1) not yolky (Fig. 3a)6 Apical cell: (0) absent; (1) present (Fig. 3c)7 Karyosome: (0) absent; (1) present (Fig. 3d)8 Occurrence of extra-DNA bodies, multiple nucleoli

and accessory nuclei: (0) absent; (1) present (Fig. 3f)9 Cortical granules: (0) absent; (1) present (Fig. 3e)10 Ovisacs, shape: (0) tubular (Figs 1d, 4b, c); (1)

spheroid (Fig. 4a)11 Ovisacs, length: (0) <1 somite long (Fig. 4a);

(1) 1–5 somites long (Fig. 4c); (2) > 5 somites long(Fig. 4b)

12 Ovisacs, convolutation: (0) absent; (1) present13 Conducting (vector) tissue: (0) absent; (1) pres-

ent (Figs 1f, 3g, h)14 Female reproductive apparatus: (0) pocket; (1)

vagina15 Female gonopore: (0) paired (Fig. 1a); (1) med-

ian (Fig. 5a)16 Copulatory area: (0) absent (Fig. 6a); (1) present

(Fig. 6b)17 Distance between gonopores (number of annuli

between the male and female gonopores): (0) <2; (1)2–3; (2) 3.5–4; (3) 4.5–5

18 Deposition of cocoons: (0) slipped off head; (1)secreted ventrally (Fig. 7a) [Note: Sawyer (1986) sug-gested that in all leeches cocoons are slipped-off. Asexamples he mentioned that Theromyzon tessulatumhas gland cells in dorsal and ventral epithelium of

reproductive segments, and thus cocoon is secretedexternally and slipped off the head in the typical way.However, in other Glossiphoniidae, e.g. Haementeriaghilianii, a clitellum is limited to ventral epithelium. Inthis case the cocoon forms near the female gonoporeand is not slipped off the head.]19 Cocoons, covering: (0) hardened shell (Fig. 7c);

(1) spongy (Fig. 7d); (2) membranous (Fig. 7b)20 Cocoons, parental care: (0) abandoned; (1)

brooded (Fig. 7a)21 Cocoons, cementation to a substrat: (0) absent;

(1) present (Fig. 1b)22 Cocoons, number of eggs per cocoon: (0) one

(Fig. 7c); (1) several [6–8]; (2) dozens (Fig. 7a, b)An additional 27 morphological characters (charac-

ters 23–49 in Table 1) were used in the analysis afterverifying them with Sereno’s (2007) criteria. We agreethat characters should be considered as independent asa critical assumption a priori for parsimony analysis.Many characters that have been used in the phyloge-netic history of Hirudinida were verified without deter-mination that they are independent and whetherneomorphic and transformative schemes are confused(Sereno, 2007). Two examples of incorrectly formu-lated character statements with our comments andsuggestions of correction according to Sereno (2007)are:1 Body shape: (0) flattened, distinctly divided into

trachelosome and urosome; (1) cylindrical, indistinctlydivided into trachelosome and urosome (Utevsky andTrontelj, 2004 after Sawyer, 1986; Epstein, 1987)—

Table 1Morphological character and state data matrix

1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 41Taxon 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9

Acanthobdella peledinaBranchiobdella parasitaBranchiobdella pentodontaGlossiphonia complanataAlboglossiphonia heteroclitaHelobdella stagnalisPiscicola geometraPiscicola margaritaePiscicola sp. Cystobranchus respiransHirudo medicinalisHirudo troctinaHaemopis sanguisugaLimnatis niloticaErpobdella octoculataErpobdella johanssoni

1 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 2 0 0 0 1 1 0 0 2 0 0 1 0 – 1 – – 0 – 1 0 0 0 – 0 0 0 0 0 0 0 – 10 0 – 0 0 0 0 0 0 – – – 0 – 0 ? 0 ? 0 0 1 0 1 0 0 1 – – 0 0 0 1 0 0 – 0 0 0 1 0 ? 1 – – 0 0 0 – 00 0 – 0 0 0 0 0 0 – – – 0 – 0 ? 0 ? 0 0 1 0 1 0 0 1 – – 0 0 0 1 0 0 – 0 0 0 1 0 ? 1 – – 0 0 0 – 01 0 0 1 0 0 1 1 1 0 1 0 0 0 1 0 1 1 2 1 – 2 1 1 1 0 1 1 0 1 1 – – 1 0 1 1 1 0 – 0 0 0 2 0 0 0 0 11 0 0 1 0 0 1 1 1 0 1 0 0 0 1 0 – 1 2 1 – 2 1 1 1 0 1 1 0 1 1 – – 1 0 1 1 1 0 – 0 0 0 2 0 0 0 0 11 0 0 1 0 0 1 1 1 0 1 0 0 0 1 0 1 1 2 1 – 2 1 1 1 0 1 0 0 1 1 – – 1 0 1 1 1 0 – 0 0 0 2 0 0 0 0 11 0 1 1 1 0 0 0 1 0 1 0 1 0 1 1 3 0 0 0 1 0 1 0 5 1 1 0 1 0 1 – – 1 1 1 0 1 0 – 0 1 0 2 0 0 1 1 21 0 1 1 1 0 0 0 1 0 1 0 1 0 1 1 3 0 0 0 1 0 1 0 5 0 1 0 1 0 1 – – 1 1 1 0 1 0 – 0 1 0 2 0 0 1 1 21 0 1 1 1 0 0 0 1 0 1 0 1 0 1 1 3 0 0 0 1 0 1 0 5 1 1 0 1 0 1 – – 1 1 1 0 1 0 – 0 1 0 2 0 0 1 1 21 0 1 1 1 0 0 0 1 0 1 0 1 0 1 1 3 0 0 0 1 0 1 1 4 1 1 0 1 0 1 – – 1 1 1 0 1 0 – 0 1 0 2 0 0 1 1 21 0 0 0 1 1 0 0 0 1 0 0 0 1 1 0 3 0 1 0 0 1 1 0 3 0 0 3 0 0 0 1 2 1 2 1 0 0 1 1 1 1 1 4 1 1 1 1 21 0 0 0 1 1 0 0 0 1 0 0 0 1 1 0 3 0 1 0 0 1 1 0 3 0 0 3 0 0 0 1 2 1 2 1 0 0 1 1 1 1 1 4 1 1 1 1 21 0 0 0 1 1 0 0 0 1 0 0 0 1 1 0 3 0 1 0 0 1 1 0 3 0 0 3 0 0 0 1 2 0 – 0 0 0 1 1 1 1 1 4 1 1 1 1 11 0 0 0 1 1 0 0 0 1 0 0 0 1 1 0 3 0 1 0 0 1 1 0 3 0 0 3 0 0 0 1 1 1 2 1 0 0 1 0 1 1 0 3 1 1 1 1 21 1 0 0 1 1 1 0 ? 0 2 1 0 0 1 0 1 0 0 0 1 1 1 0 3 0 0 2 0 0 0 0 – 0 – 0 0 1 0 – 0 0 0 5 1 0 1 1 11 1 0 0 1 1 1 0 ? 0 2 1 0 0 1 0 1 0 0 0 1 1 1 0 3 0 0 2 0 0 0 0 – 0 – 0 0 1 0 – 0 0 0 5 1 0 1 1 1

‘–’ not applicable; ‘?’ unknown or uncertain.

A. Bielecki et al. / Cladistics 30 (2014) 540–554 543

independent character states were used as dependentstates; division of this character statement allows usto define the correct determined variable (body shape,division on trachelosome and urosome).

2 Somatic sense organs (after Utevsky and Trontelj,2004)/body ornamentation (after Williams and Burre-son, 2006): (0) absent; (1) papillae; (2) tubercles—neo-morphic and transformative schemes were confused;the character state “absent” was used incorrectly(“absent” is not a sense organ).Characters are adopted from Apakupakul et al.

(1999), Borda and Siddall (2004a), Siddall and Bur-reson (1995), and Williams and Burreson (2006).After verification following ‘not-female’ morphologi-cal characters were used for parsimony analysis:

23 Setae: (0) present; (1) absent

24 Body shape: (0) vermiform; (1) dorsoventrallyflattened25 Annuli per somite: (0) 2; (1) 3; (2) 4; (3) 5; (4) 7;

(5) 1426 Annuli in somite: (0) equal; (1) unequal27 Eyes, arrangement: (0) dorsolateral; (1) dorsal.28 Eyes, number: (0) 2 pairs; (1) 3 pairs; (2) 4 pairs;

(3) 5 pairs29 Pulsative vesicles: (0) absent; (1) present30 Mouthpore, location: (0) central; (1) terminal31 Pharynx: (0) not protrusible; (1) protrusible pro-

boscis32 Myognaths: (0) unarmed; (1) armed33 Denticles, number: (0) few; (1) about 30; (2)

about 6034 Crop cecae: (0) absent; (1) present

a d

b

c

e

f

Fig. 1. Details of ovary organization and oogenesis in branchiobdellidans and Acanthobdella peledina. (a) Branchiobdella pentodonta, with malegonopore (I), female gonopores (II), spermathecal pore (III), and part of posterior adhesive disc (ad) marked. SEM. Scale bar = 60lm. (b)B. pentodonta: cocoons with peduncles (arrows) cementing them to a substrate. SEM. Scale bar = 25 lm. (c) Branchiobdella parasita: the ovaryis a conically shaped structure, connected to the intersegmental septum (is) by a thin ligament (l); inside the ovary, germ cells (g), cytophore (cy),and somatic cells (arrow) are visible. Body cavity (bc). LM, epon semi-thin section stained with methylene blue. Scale bar = 25 lm. (d) A. peledi-na: only one elongated ovary cord (encircled) is enclosed inside each ovisac (os), which passes directly into the oviduct (ov). FM, whole-mountpreparation of ovaries stained with DAPI. Scale bar = 250 lm. (e) A. peledina: inside the ovary sac (os) details of the ovary cord organizationare visible; germ cells (gc) are connected to the well-developed cytophore (cy), and between them somatic cells (sc) occur. LM, epon semi-thinsection stained with methylene blue. Scale bar = 40 lm. (f) A portion of the cross section through segment XIII of the A. peledina body: bodywall (bw), vector tissue (vt), oviduct (ov), nerve cord (nc), midgut lumen with blood (mg), muscles (ms) and ventral blood vessel (vbv) are visible.LM, epon semi-thin section stained with methylene blue. Scale bar = 80 lm.

544 A. Bielecki et al. / Cladistics 30 (2014) 540–554

35 Crop cecae, number: (0) 6 pairs; (1) 7 pairs; (2)10 pairs

36 Postcecae: (0) absent; (1) present37 Intestine, cecae: (0) absent; (1) present38 Male bursa: (0) single bulb; (1) bilobed39 Penial apparatus: (0) absent; (1) present [Note:

in the genus Theromyzon this structure is protrusible,and in some representatives of the family Piscicolidaeit is eversible, but it is not homologous with a penis ofthe Arhynchobdellida.]

40 Penis, shape: (0) straight; (1) recurved41 Epididymes: (0) loose; (1) tightly coiled42 Accessory glands: (0) absent; (1) present43 Ejaculatory ducts: (0) U-shaped; (1) S-shaped44 Testisacs, number: (0) 1 pair; (1) 2 pairs; (2) 6

pairs; (3) 8 pairs; (4) 10 pairs; (5) > 10 pairs45 Nephridia: (0) complete in genital segments; (1)

suppressed in genital segments46 Urinal bladder: (0) absent; (1) present47 Dorsal and lateral lacunae, connection: (0)

absent; (1) present48 Muscularization of lateral coelomic spaces: (0)

absent; (1) present.

49 Feeding strategy: (0) commensalism; (1) preda-tory; (2) sanguivorous

Phylogenetic analyses

Parsimony analyses were performed using PAUP*4.06b10 for Windows, for IBM PC (Swofford, 2000)using the branch-and-bound option for both charac-ters of the female reproductive system separately aswell as for the combined data set. All characters wereparsimony informative, unordered, and of equalweight. Statistical support for the putative clades wasassessed using bootstrap analysis (200 replicates) underthe heuristic search option with random taxon addi-tion and TBR branch swapping. Acanthobdella peledi-na, Branchiobdella parasita, and B. pentodonta wereconsidered as outgroup taxa. All trees were exploredin FigTree version 1.3.1 (Rambout, 2009). Evolutionof selected characters was traced using Mesquite ver-sion 2.75 (Maddison and Maddison, 2011) under theoption trace character history.

a b

c d

e f

Fig. 2. (a–d) General organization of the leech ovary. (a) Piscicola geometra: numerous egg follicles develop within the ovisac. Scale bar = 70 lm.(b) Alboglossiphonia heteroclita: one, huge and convoluted ovary cord within the ovisac. Scale bar = 125 lm. (c) Erpobdella octoculata: one of theseveral shortened ovary cords occurring in each ovisac. Scale bar = 150 lm. (d) Haemopis sanguisuga: one of two long and convoluted ovary cordsobserved within each ovisac. Arrows mark developing oocytes. Scale bar = 350 lm. (e, f) Cytophore organization. (e) E. octoculata: a cytophorehas a form of thin cytoplasmic strands. Scale bar = 30 lm. (f) A. heteroclita: a cytophore is huge and occupies the centre of the ovary cord. Scalebar = 25 lm. cy, cytophore; ef, egg follicles; nc, nurse cells; o, developing oocytes; ovc, ovary cord; ow, ovary wall.

A. Bielecki et al. / Cladistics 30 (2014) 540–554 545

Results

Parsimony analysis based on characters concerningovary structure and oogenesis of leeches alone (22characters) resulted in two equally most-parsimonioustrees (length = 30, CI = 0.90, RI = 0.96). The strict

consensus of these trees (Fig. 8) resolved six clades outof a total 13 possible for 15 taxa. The tree shows bothRhynchobdellida and Arhynchobdellida as monophy-letic. Piscicolidae appeared to be sister to Glossiphonii-dae, whereas Erpobdellidae appeared to be sister toHirudinidae + Limnatis nilotica. Neighbour-joining

a b

c d

e f

g h

Fig. 3. (a–f) Details of ovary organization and oogenesis in leeches. (a) Piscicola geometra; a poorly equipped yolk oocyte arrested in meioticmetaphase I; arrow marks chromosomes. Scale bar = 15 lm. (b) Alboglossiphonia heteroclita: a yolk-rich oocyte during late oogenesis (vitelogen-esis); arrow points to oocyte nucleus. Scale bar = 70 lm. (c) Hirudo medicinalis: an apical cell at the tip of ovary cord. Scale bar = 15 lm. (d)Helobdella stagnalis: an oocyte nucleus in late oogenesis, and within the nucleus the karyosome is visible; nuclear bodies are marked by arrows.Scale bar = 15 lm. (e) P. geometra: cortical granules (arrows) occur in the peripheral cytoplasm. Scale bar = 15 lm. (f) A. heteroclita: in closeproximity of the oocyte nucleus an accessory nucleus is visible. Scale bar = 2 lm. (g, h) Vector tissue in P. geometra. (G) Vector tissue lies belowthe body musculature in the body region termed the copulatory area (see Fig. 5). Scale bar = 180 lm. (h) Details of vector tissue ultrastructure:two kinds of vector tissue cells are visible, i.e. vesicular and granular cells. Scale bar = 15 lm. an, accessory nucleus; ap, apical cell; gc, granularcells; k, karyosome; m, muscles; nu, oocyte nucleus; o, oocyte; vc, vesicular cell; vt, vector tissue; y, yolk.

546 A. Bielecki et al. / Cladistics 30 (2014) 540–554

analysis supported the parsimony results (data notshown). Species of the taxa Piscicolidae and Hirudini-formes form a polytomic clade each. Two additionalsteps were needed to make rhynchobdellids paraphylet-ic and render a topology in concordance with the clad-ogram resulting from the latest studies on themolecular level (e.g. Siddall et al., 2001).

Use of combined data (22 female and 27 other mor-phological characters) also resulted in four most-parsi-monious trees (83 steps, CI = 0.81, RI = 0.91). Thestrict consensus of these trees (Fig. 9) resolved eightclades out of a total 13 possible for 15 taxa. This“total evidence” cladogram shows the same result asgiven above, but with the polytomies within Piscicoli-dae and Hirudiniformes being somewhat moreresolved. Piscicola margaritae is sister to the polytomicclade of P. geometra + P. sp. + Cystobranchus respi-rans. Within Hirudiniformes Haemopis sanquisuga issister to the polytomic clade of Hirudo medicinalis +H. troctina + L. nilotica. Four extra steps wererequired if forcing the topology to be like recentmolecular results (e.g. Siddall et al., 2001).

Discussion

This study allows for the interpretation of the polar-ity of the ultrastructural features of the female repro-ductive system in a cladistic context for the first time(characters 1–9). The course of oogenesis within theovisacs (char. 1: 1) and organization of the ovarian

a b

Fig. 5. Female gonopore. (a) Erpobdella vilnensis: female gonopore(arrow) is present. Scale bar = 0.25 cm. (b) Alboglossiphonia hyalina:female gonopore is absent. Scale bar = 0.66 cm.

a b

Fig. 6. Copulatory area. (a) Cystobranchus mammillatus: copulatory area is lacking. Scale bar = 0.60 cm. (b) Piscicola siddalli: copulatory area ispresent (arrow). Scale bar = 0.10 cm.

a b c

Fig. 4. Ovisacs. (a) Limnatis nilotica: short, spheroid, half-somite long ovary (arrow). Scale bar = 0.25 cm. (b) Erpobdella testacea: a tubularovary more than three somites long (arrow). Scale bar = 0.75 cm. (c) Piscicola brylinskae: a tubular ovary less than two somites long (arrow).Scale bar = 0.35 cm.

A. Bielecki et al. / Cladistics 30 (2014) 540–554 547

internal units as ovary cords (char. 3: 0) seem to beplesiomorphies occurring in Acanthobdellida andHirudinida. This contrasts with previous work (e.g.Marotta et al., 2008), suggesting a closer relationshipof acanthobdellids, rather than branchiobdellidans, tohirudinids. In that case occurrence of ovisacs andovary cords would be synapomorphies for chaetiformand true leeches. Non-polarized ovary cords (char. 4:1) and presence of cortical granules in ooplasm (char.9: 1) appeared to be synapomorphies in rhynchobdellidleeches (Fig. 10a,b). These characters may suggest themonophyly of Rhynchobdellida, a fundamental findingthat is in conflict with results of the latest phylogeneticanalyses based on molecular and morphological data(Siddall and Burreson, 1998; Apakupakul et al., 1999).In Arhynchobdellida there is one synapomorphy affor-ded by the female reproductive data, namely the pres-ence of an apical cell (char. 6: 1; Fig. 10c), whichsomewhat confirms the monophyly of the groupshown by other researchers (Apakupakul et al., 1999;Borda and Siddall, 2004b). Lack of yolk in eggs (char.

5: 1) and presence of a karyosome (char. 7: 1) bothappear to be homoplasious (reversals) (Fig. 11a,b).The poorly developed cytophore (char. 2: 1) inErpobdella, presence of egg follicles (char. 3: 1) in Pisci-colidae, and occurrence of extra-DNA bodies, multiplenucleoli, and accessory nuclei (char. 6: 1) in Glossipho-niidae all seem to be autapomorphies, useful perhapsfor family-group membership but of limited utility inthe higher-level analyses of phylogenetic relationships.The most-parsimonious cladogram based on 22

characters associated with the female reproductive sys-tem and oogenesis details when taken alone (Fig. 8)exhibits the ingroup as three monophyletic groups ofBranchiobdellida, Glossiphoniidae + Piscicolidae, andHirudiniformes + Erpobdellidae. The last of these (i.e.the monophyly of Arhynchobdellida) corroborates pre-vious findings at the morphological and molecularlevel (Trontelj et al., 1999; Siddall et al., 2001; Jamie-son et al., 2002; Utevsky and Trontelj, 2004). Intrigu-ingly, our results suggest the need to reconsider themonophyly of Rhynchobdellida, a group present in

a

b c d

Fig. 7. Cocoons. (a) Placobdella costata: hundreds of eggs in a cocoon secreted ventrally. Scale bar = 0.23 cm. (b) Glossiphonia verrucata: dozensof eggs in cocoons without spongy covering. Scale bar = 0.50 cm. (c) Piscicola geometra: one egg in a cocoon. Scale bar = 0.40 cm. (d) Hirudomedicinalis: cocoon with spongy covering. Scale bar = 0.33 cm.

548 A. Bielecki et al. / Cladistics 30 (2014) 540–554

traditional classifications (e.g. Mann, 1962; Sawyer,1986), and yet not emergent in molecular phylogeniesof leech groups.In Hirudinida studied so far the organization of

ovaries shows a diversity that can be generalized asthree systems of internal ovary organization. Briefly,polarized ovary cords occur in Hirudinidae, Haemopi-dae, and Erpobdellidae, non-polarized ovary cords

were found in ovaries of Glossiphoniidae, and finallyegg follicles were found in Piscicolidae (for details see�Swiaztek, 2008; Urbisz et al., 2010). Which system isbasal, and which is the most advanced? Our knowl-edge of the ovary organization in the closest euhirudi-nean relatives (i.e. Branchiobdellidae, Acanthobdellida,and Lumbriculida, see �Swiaztek et al., 2012; Urbisz and�Swiaztek, 2013) and observations made by Livanow

Fig. 8. The most-parsimonious tree for 13 leech species based on data concerning ovary structure and course of oogenesis. Acanthobdella peledi-na, Branchiobdella parasita, and B. pentodonta were chosen as an outgroup. Numbers on the nodes indicate boostrap values.

Fig. 9. The most-parsimonious tree for 13 leech species based on combined data sets of 22 characters of the ovary structure and course ofoogenesis and 27 non-reproductive morphological characters. Acanthobdella peledina, Branchiobdella parasita, and B. pentodonta were chosen asan outgroup. Numbers indicate boostrap values.

A. Bielecki et al. / Cladistics 30 (2014) 540–554 549

(1906) on A. peledina suggest that the polarized ovarycord found in Hirudinidae and Limnatis nilotica(a representative of the family Semiscolecidae), andshortened ovary cords found in Erpobdellidae can beregarded as the original condition for Hirudinida. Inboth Hirudinidae + L. nilotica and Erpobdellidae thereis an apical cell at the tip of the ovary cord, and alongthe long axis of the cord the consecutive stages ofoogenesis can be observed (polarization of the cord)(J€orgensen, 1908; Van Damme, 1974; �Swiaztek, 2008;Ben Ahmed et al., 2010, 2013; �Swiaztek et al., 2010a).Both features (polarized ovary cords and the apical

cell) indicate a close phylogenetic relationship betweenErpobdelliformes and Hirudiniformes shown previ-ously in studies based on molecular and morphologicaldata (Trontelj et al., 1999; Siddall et al., 2001; Jamie-son et al., 2002).According to Borda and Siddall (2004a,b; see also

Siddall and Burreson, 1996; Rousset et al., 2008) theancestral leech was adapted to the freshwater environ-ment, supported by the shared ancestry of the leecheswith freshwater Acanthobdellida, Branchiobdellida,and Lumbriculida. Siddall and Burreson (1996) suggestthat the aquatic habits of erpobdellids represent a sec-

a

b

Fig. 10. Evolution of chosen female reproductive characters inleeches. (a) Polarization of ovaries. (b) Apical cell.

a

b

Fig. 11. Evolution of chosen female reproductive characters inleeches. (a) Eggs. (b) Karyosome.

550 A. Bielecki et al. / Cladistics 30 (2014) 540–554

ondary adaptation to a freshwater lifestyle. The aqua-tic “proto-leech” laid hard-shell cocoons on a solidsubstrate (smooth stones, etc.); this type of behaviouris evident in aquatic erpobdellids and has been shownin the chaetiferous leech-like worm A. peledina (Kutsc-hera and Wirtz, 1986, 2001; Sawyer, 1986; Pfeifferet al., 2005; Rousset et al., 2008).The ovary organization and the course of oogenesis

in Glossiphoniidae and Piscicolidae are essentially dif-ferent (Spałek-Wołczy�nska et al., 2008). Both systemsshould be regarded as derived. In glossiphoniids thereremain ovary cords, but no apical cell has been foundand the cords are non-polarized; oogenesis is synchro-nous and at a certain point we can observe only young(previtellogenic) or late (vitellogenic) oocytes (Fern�an-dez et al., 1992; �Swiaztek, 2005a,b, 2006). Additionally,glossiphoniids produce dozens or even hundreds of yo-lky eggs. The most unusual system of ovary organiza-tion was found in piscicolids. There are no ovarycords in these leeches, but instead each cyst of germcells is surrounded by its own group of somatic cellsand forms an egg follicle (Fischer and Weigelt, 1975;Spałek-Wołczy�nska et al., 2008). Furthermore, Pisci-colidae lay only one, yolk-less egg per cocoon (fordetailed comparison of ovary structure in Glossipho-niidae and Piscicolidae see Spałek-Wołczy�nska et al.,2008). The mutually and differently derived conditionsare reflected in the low value of branch support (boo-strap value = 57). Nonetheless, there are some rhync-hobdellid synapomorphies—including non-polarizedovary cords, presence of cortical granules [structuresinterpreted as cortical granules have been found insome erpobdellids (Ben Ahmed et al., 2013), althoughtheir molecular composition and function areunknown, and thus in our analyses the feature wastreated as uncertain] and ovisacs of 1–5 neurosomites(ns) in length—that support the monophyly of rhync-hobdellid leeches shown on the cladogram obtained(Fig. 10a,b).Vector tissue is an important feature of the female

reproductive system (Figs 1f, 3g,h), particularly in thepiscolids. Vector tissue was found and described inAcanthobdella peledina (Epstein, 1987) and piscicolidleeches only (Brumpt, 1900; Sawyer, 1986; �Swiazteket al., 2007, 2010b; Fig. 1f); however, there are somesuggestions that vector tissue occurs also in representa-tives of Glossiphoniidae (Brumpt, 1900; Bielecki,2004). Greater attention to these morphological char-acters in future anatomical studies of leeches shouldclarify the homology of these structures across groups.Ovarian length entails many problems of interpreta-

tion, because of the poor studied comparative anat-omy, principally in the family Glossiphoniidae.However, newly undertaken examinations shouldallow proper interpretation of this feature. We still donot know the variability of the length of ovaries in

Hirudiniformes, but it appears that their lengthcorresponds to half a somite (A. Bielecki and J.M. Ci-chocka, unpublished data; Fig. 4a).A “copulatory area” appearing in leeches used in

analysis belonging to the family Piscicolidae is a recog-nizable apomorphic feature (Fig. 6b), but there arespecies, including freshwater piscicolids, for which thisfeature is lacking, as is also the case in marine piscico-lids (Utevsky and Trontelj, 2004; Utevsky, 2007). Atpresent, the copulatory area appearing in Cystobran-chus respirans, Baicalobdella torquata (Grube, 1871),and Piscicola geometra seems to be a convergent char-acter rather than an apomorphic one (Epstein, 2004;Utevsky and Trontelj, 2004).A separate female gonopore is an extraordinary fea-

ture that is also known in species of the genus Ozobran-chus (Siddall and Burreson, 1995). Species that possesspaired female gonopores in the present study includeBranchiobdella sp. (Fig. 1a) and Theromyzon maculosum(Sawyer, 1986; Brinkhurst and Gelder, 1989). These spe-cies have more in common in terms of reproductivecharacters, for example lack of a fused oviduct orvagina and the presence of a penial apparatus (penis)(Sawyer, 1986). It appears that those characters areplesiomorphies or homoplasies. Only A. heteroclita doesnot have a separate female gonopore at all (Fig. 5b).Our analyses based on characters of the female

reproductive system separately show Erpobdelliformesas sister to Hirudiniformes (boostrap value = 61;Fig. 8). This result confirms the results obtained inmolecular studies (Trontelj et al., 1999; Siddall et al.,2001; Jamieson et al., 2002; Oceguera-Figueroa et al.,2011; Nakano et al., 2012) as well as phylogeneticinferences based on sperm structure (Ferraguti andErs�eus, 1999; Marotta et al., 2008). However, the addi-tional presence of several homoplasies within thefemale reproductive system in Arhynchobdellida mayexplain the overall low value of branch support.Taken together, we believe that the characters con-

cerning the structure of the female reproductive systemand course of oogenesis are conservative and that onthe whole they well reflect phylogenetic relationshipsof Hirudinida on the family level (Fig. 8).Our analyses based on the combined data set

(Fig. 9) yielded the same result in terms of generaltopology, but some within-family polytomies were par-tially resolved. Within piscicolids the genus Piscicolaappeared to be non-monophyletic because of the place-ment of C. respirans; only one additional step isrequired to make Piscicola monophyletic. That beingsaid, the position of C. respirans is not without histori-cal complications. According to Epstein (1969) andBielecki (1997), the species should actually be includedin the genus Piscicola. Our present results corroboratethis idea. However, both Sawyer (1986) as well asmolecular studies by Utevsky and Trontelj (2004) and

A. Bielecki et al. / Cladistics 30 (2014) 540–554 551

Utevsky (2007) imply a distinct genus for members ofCystobranchus.Prior phylogenetic investigations of Arhynchobdell-

ida have questioned the monophyly of traditionalfamilies, especially within Hirudiniformes (Siddall andBurreson, 1995; Apakupakul et al., 1999; Tronteljet al., 1999; Borda and Siddall, 2004a,b; Borda et al.,2008; Phillips and Siddall, 2009). Our results supportthe monophyly of the family Hirudinidae as shown byPhillips and Siddall (2009), as a taxonomic group con-taining sanguivorous Hirudo and predatory Haemopis.Limnatis nilotica, although it is a blood-sucking leechof the Old World, did not form a monophyletic cladewith other examined haematophags (Fig. 9).The characters of the female reproductive system

and other morphological features (e.g. body shape,body ornamentation, annulation, eyes and ocelli, struc-ture of the alimentary tract, structure of the malereproductive system, behaviour related to reproduc-tion) are usually described in a manner as to showprobable phylogenetic reconstruction of leeches on alower level than family. The cladogram (Fig. 9)resolves polytomic sets of genera and species withinfamilies. Thus, when we discuss phylogenetic relation-ships, we use names of the leech families, because thestructure of the female reproductive system and courseof oogenesis is characteristic for the family level.Recognizing that there remain issues concerning the

origin of leeches, we examined two distinct outgrouptaxa for polarization: with Acanthobdella peledina inthe outgroup only, and with Branchiobdella sp. only asthe outgroup. The first results in the same topology as

that obtained using all outgroup taxa. However, usingonly female reproductive characters and only Branch-iobdella sp. as an outgroup taxa the result was onlytwo most-parsimonious trees (length = 29, CI = 0.90,RI = 0.96; Fig. 11), the strict consensus of which dif-fered from all other approaches; Glossiphoniidae andPiscicolidae formed two distinct branches, implyingthat Rhynchobdellida is not a monophyletic group.Arhynchobdellida remain a monophyletic group withErpobdellidae sister to Hirudinidae + L. nilotica(Fig. 12).We conclude that characters concerning the female

reproductive system and course of oogenesis providehigh-quality comparative data that are useful in con-siderations of the phylogenetic reconstruction of Hiru-dinida, although perhaps only at the family level.Additional character sets could similarly benefit fromsuch analyses in a cladistic context. These include, forexample, models of the leech body form (Bielecki andEpstein, 1994; Bielecki, 1997; Bielecki et al., 2009,2011b, 2012, 2013), new external and internal morpho-logical characters, and molecular and other availablecomparative data (biogeographical, biotopical,behavioural diversity, information from the parasite–host systems, etc.).

Acknowledgements

This work was partially supported by research grantN303 423636 from the Polish Ministry of Science andHigher Education. We are grateful to Dr Witold

Fig. 12. The most-parsimonious tree for 13 leech species based on data concerning ovary structure and course of oogenesis. Branchiobdella para-sita and B. pentodonta were chosen as an outgroup. Numbers on the branches indicate boostrap values.

552 A. Bielecki et al. / Cladistics 30 (2014) 540–554

Stru _zy�nski, Division of Zoology, Faculty of AnimalSciences, Warsaw University of Life Sciences SGGW,Warsaw, Poland, for collecting branchiobdellids, toRaja Ben Ahmed, Universit�e de Tunis El-Manar,Facult�e des sciences de Tunis, Tunisia, for collectingHirudo troctina, Limnatis nilotica, and Erpobdellajohanssoni. B.J.P. gratefully acknowledges a scholarshipfor Outstanding Young Scientists from the Minister ofScience and Higher Education.

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