Do circum-Antarctic species exist in peracarid Amphipoda? A case study in the genus Epimeria Costa, 1851 (Crustacea, Peracarida, Epimeriidae)

Th e amphipod genus Epimeria is species rich in the Southern Ocean and at present eight of its 19 species are reported with circum-Antarctic distributions. For the fi rst time, specimens of epimeriid species from the Antarctic Peninsula, the Weddell Sea and the Ross Sea were analysed using partial COI genes sequences and morphological characters. In total 37 specimens of 14 species of Epimeria and two species of Epimeriella were analysed and the resulting molecular topology checked by critically reviewing taxonomic characters. Th e genus Epimeriella, genetically grouping within Epimeria is synonymised with the genus Epimeria. Sequences distances between populations of the nominal species Epimeria robusta from the Weddell and Ross Sea led to detailed morphological investigations, resulting in the description of Epimeria robustoides sp. n. from the Weddell Sea. Epimeria robusta Barnard, 1930 from the Ross Sea is redescribed. Sequences of a damaged Epimeria specimen of a species new to science from the lower continental shelf of the eastern Weddell Sea were included. Based on the current study, the hypothesis of circum-Antarctic species’ distributions in brooding amphipods proved to be unlikely. ZooKeys 18: 91–128 (2009) doi: 10.3897/zookeys.18.103 www.pensoftonline.net/zookeys Copyright Anne-Nina Lörz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Launched to accelerate biodiversity research A peer-reviewed open-access journal


Introduction
In the Southern Ocean's benthic ecosystem, crustaceans are by far the most specious taxon.Among the crustaceans, amphipods are the most numerous group with more than 815 recorded species (De Broyer et al. 2007).Th e globally distributed amphipod family Epimeriidae Boeck, 1871 (formerly Paramphithoidae) belongs to the dominant members of Antarctic shelf benthos (Coleman 2007).Twenty-fi ve species of Epimeriidae are known from Antarctic waters, that is 19 Epimeria Costa, 1851, four Epimeriella Walker, 1906, one Metepimeria Schellenberg, 1931a and one Uschakoviella Gurjanova, 1955b.
Based on their distribution records from the Weddell and Ross Sea shelves, eight species of Epimeriidae (Epimeria grandirostris, E. inermis, E. macrodonta, E. puncticulata, E. "robusta", E. macronyx, E. walkeri and E. scabrosa) are believed to have circum-Antarctic distributions.Epimeria robusta Barnard, 1930 was originally described from the Ross Sea but because of insuffi cient descriptions and images Coleman (1994) redescribed E. robusta based on specimens from the Weddell Sea.Five of the epimeriid species (Epimeria extensa, E. heldi, E. reoproi, E. vaderi and E. truncata) are known from locations only on the Antarctic Peninsula.Two species, Epimeria rimicarinata Watling and Holman, 1980 and the recently described E. schiaparelli Lörz, Maas, Linse and Fenwick, 2007 are found exclusively in the Ross Sea.
Epimeriella macronyx is known from the eastern Antarctic Peninsula, Davis Sea, Ross Sea, South Orkney Islands, South Shetland Islands and the Weddell Sea.Epimeriella scabrosa was found at Oats Coast and the Weddell Sea.Epimeriella truncata is known only from the type locality, at the western Antarctic Peninsula.Epimeriella walkeri has a distribution at the Davis Sea, Palmer Archipelago, Ross Sea, South Shetland Islands and Weddell Sea.Th e monotypic genus Metepimeria has never been found again after its original description and the fi nding of Uschakoviella by Watling and Holman (1981) could not be confi rmed.All other known species of Uschakoviella are restricted to the Arctic Ocean.
Th e Antarctic Epimeriidae mostly occur on the continental shelves and upper slopes.So far only one epimeriid specimen has been caught on the deep slope (2157 m) off Kapp Norvegia in the Weddell Sea during the ANDEEP III expedition.Th is damaged species of Epimeria sp.proved to be new to science (authors personal observation).
Outside of the Southern Ocean only a few species of Epimeriidae have been described from the Southern Hemisphere.With the exception of Epimeriella victoria (Hurley, 1957) these belong to the genus Epimeria and show bathymetric affi nities to the deep continental slopes and deep sea.Th ree have been found in depths greater than 1500 m off the Brazilian coast: E. bathyalis Wakabara and Serejo, 1999(1200-1575 m), E. rotunda Wakabara and Serejo, 1999(1190-1205 m) and E. ultraspinosa Wakabara and Serejo, 1999 (830 m).Th e deepest species of Epimeriidae found is Epimeria bispinosa Ledoyer, 1986 off Madagascar in 3450 m water depth.Epimeria longispinosa Barnard, 1916 occurs off eastern Florida and False Bay, South Africa at depths of 345-750 m.Th e New Zealand Epimeriidae so far only comprises of four species [Epimeria glaucosa Barnard, 1961, 3710 m; E. bruuni Barnard, 1961Barnard, , 2470 m; m;E. horsti Lörz, 2008and E. victoria (Hurley, 1957), 140 m] and to date none have been found in Australian waters (Lörz et al. 2008).
Th e fi rst combined molecular and morphological phylogeny of Antarctic Epimeriidae and Iphimediidae was based on a total of 16 taxa.It was presented by Lörz and Held (2004) and proved the monophyly of the families Epimeriidae and Iphimediidae.Th is preliminary study based on only 16 specimens from the Weddell Sea included six species of Epimeria (Epimeriidae) and eight species of Iphimediella, Echiniphimedia and Gnathiphimedia (Iphimediidae).Lörz and Brandt (2004) published the fi rst extensive morphology-based phylogeny of Antarctic Epimeria based on all 17 Antarctic species described at that time, including species of the genera Epimeriella and Metepimeria.Th e resulting topology of this study confi rmed the monophyly of the Epimeriidae and Iphimediidae but was not convincingly able to determine relationships between and within the genus Epimeria.Epimeriella and Metepimeria species appeared amongst the species of Epimeria, suggesting polyphyly for the latter genus.
Recent expeditions to the Ross Sea, seamounts off New Zealand and the Weddell Sea collected new epimeriid material, which was preserved in a state suitable for genetic studies.Th is new material enables us is to shed light on open questions regarding the evolution of Southern Hemisphere Epimeriidae: 1. Do circum-Antarctic distributions occur amongst species of epimeriid Amphipoda?2. How are New Zealand and Antarctic Epimeria species related?Do the Southern Ocean epimeriids form an Antarctic clade? 3. Are the genera Epimeria and Epimeriella monophyletic?Our recent study contributes to the ongoing investigation and census of the Southern Ocean benthic biota, its diversity and biogeographic history.

Material and methods
Taxon sampling.During recent expeditions of RV Tangaroa to the Ross Sea (BioRoss, TAN0402; IPY, TAN0802) and seamounts off New Zealand (TAN0413, TAN0602), as well as RV Polarstern to the Weddell Sea (ANT XXI/2, BENDEX und ANDEEP III) new amphipod material was collected.Amphipods were sorted from collections immediately (often alive), fi xed in 98% ethanol and later transferred to 70% ethanol.Of these collections 30 specimens of Epimeriidae and 1 specimen of Iphimediidae were identifi ed to species level and included in the molecular analysis (Table 1).Th e fi nal phylogenetic dataset includes the 31 new sequences of 14 epimeriid and one iphimediid species and 17 published sequences of six species of Epimeriidae, nine of Iphimediidae and Eusirus cf.perdentatus (Eusiridae).
Morphological description.Specimens were examined and dissected using a Leica MZ9.5 stereomicroscope and drawn using a camera lucida attachment.Small appendages (mouthparts, uropods, telson) were temporarily mounted in lactic acid, examined and drawn using a Nikon compound microscope fi tted with a camera lucida.Th e body lengths of specimens examined were measured by tracing individual's mid-trunk lengths (tip of the rostrum to end of telson) using a camera lucida.
All illustrations were drawn by using the digital inking illustration method described by Coleman (2003).Within the description, abbreviations are used for slender setae (SS) and robust setae (RS).Type material was deposited in the Natural History Museum Berlin, Germany, and the NIWA Marine Invertebrate Collection Wellington, New Zealand.We cross checked with the type material from E. robusta, held at the Natural History Museum London (BMNH 1930.8.1.303-309).Coloured photographs of Epimeria robusta and E. robustoides sp.n. were taken on board immediately after the specimen were caught.
DNA extraction and analysis.Genomic DNA was isolated from amphipod pereopods using the DNEasy tissue extraction kit (Qiagen Ltd) and quantifi ed using the PicoGreen quantifi cation kit (Molecular Probes, Invitrogen Ltd).Th e partial mitochondrial cytochrome oxidase subunit I (COI) gene was amplifi ed using the universal primers described by Folmer et al. 1994 using PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare), 0.2 μM of each primer and between 20-200 ng of genomic DNA.PCR reactions were carried out in a GeneAmp 2720 thermocycler (Applied Biosystems, Foster City, California, USA) using the following conditions: an initial hold at 95°C for 5 minutes and then 30 cycles of 95°C for 30 seconds; 45°C for 30 seconds; 72°C for 1.5 minutes; and a fi nal extension at 72°C for 7 minutes.PCR products were purifi ed using QIAquick Spin Columns (Qiagen Ltd) and quantifi ed using the PicoGreen Kit (Molecular Probes, Invitrogen Ltd).Sequencing of the COI gene was carried out at Macrogen Ltd, Korea, using the amplifi cation primers.
Th e proof-read sequences of the 31 specimens were aligned using ARB software (Ludwig et al. 2004) against COI sequences available in EMBL of seven Epimeria species, three Echiniphimedia species, three Iphimediella species, two Gnathiphimedia species and Eusirus cf.perdentatus Chevreux, 1912 (Table 1).Th e Iphimediidae and Eusirus cf.perdentatus were chosen as the outgroup taxa, since Lörz and Held (2004) showed them to be the sister taxa of the Epimeriidae.
Evolutionary distances were calculated from sequence pair dissimilarities using only unambiguously sequenced positions.Th e partial COI gene sequences determined in this study are deposited in the EMBL database and the accession number for each specimen is shown in Table 1.Th e amphipod specimens are registered and curated at the National Institute for Water and Atmospheric Research (NIWA) (Table 1).
Phylogenetic analysis.Sequences were analyzed using maximum parsimony (MP) and maximum likelihood (ML) criteria in PAUP*4.0b10(Swoff ord 2002).MP analyses were implemented in PAUP* as heuristic search, tree bisection-reconnection (TBR), random addition sequence.All characters were unordered and analyses were conducted under equal weights.Topological robustness was assessed using 1000 bootstrap replicates (Felsenstein 1985) and parsimony jackknifi ng (Farris et al. 1996).Jackknife frequencies were calculated in PAUP* using 1000 pseudoreplicates under a heuristic search with 30% character deletion.Th e ML analysis used the HKY85 model.Th e ML analysis was conducted using the heuristic search option in PAUP* and starting branch lengths were obtained using Rogers-Swoff ord approximation method.Bootstrap values for the ML tree were obtained from 100 replicates.Morphological descriptions.Th e taxonomic diff erentiation within and between genera of the Epimeriidae is often based on a few morphological characters (Coleman and Barndard 1991, Lörz and Brandt 2004).A key to the Antarctic species of Epimeriidae was published by Coleman (2007).Th e morphological characterisation in Epimeriidae is hindered by the high plasticity of characters depending on sex and age of the specimens (Lörz and Brandt 2004), therefore taxon specifi c characteristics can the misinterpreted as intraspecifi c variability.Genetic information, here COI mtDNA, is used as additional characters to clarify and validate the taxonomic classifi cation (Figure 1).
Here  2007), Coleman and Barnard (1991), and Barnard and Karaman (1991).Based on the genetic data presented in the following and a detailed morphological evaluation of the weak morphological separation criteria between Epimeriella and Epimeria we herewith synonymize Epimeriella with Epimeria.Since we transfer the species Epimeriella macronyx Walker, 1906; Epimeriella scabrosa Barnard, 1930;Epimeriella truncata Andres, 1985;Epimeriella victoria (Hurley, 1957a) and Epimeriella walkeri Barnard, 1930 to the genus Epimeria, we herewith give a new genus diagnosis.
Th e diagnosis has been broadened from that given by Barnard and Karaman (1991) since it now included characters formerly predominantly occurring in the genus Epimeriella such as a smooth body and the laminar, none triturative pars molaris.
Urosome and telson.Uropod 1: peduncle subequal in length to inner ramus, medial margin with 1 RS distally, distal margin with close row of short RS; inner ramus lateral margin with spaced row of short RS, medial margin with sparse RS; outer ramus marginally shorter than inner.Uropod 2: peduncle with row of short setae; inner ramus nearly twice the length outer ramus, both margins sparse lined with RS; outer ramus, both margins with few short RS.Uropod 3: peduncle short, approx.0.3 × length of inner ramus, medial and inner margins of both rami with sparse row of short RS.Telson slightly longer than wide, u-shaped emargination 0.2 × lengths, lobes triangular, broadly rounded apically.
Coloration.Freshly captured specimen (s) of Epimeria robustoides show distinct red eyes (Fig. 10 A) and some bear orange patches on their bodies.
Urosome and telson.Uropod 1: peduncle subequal in length to inner ramus, medial margin with 1 RS distally, distal margin with close row of short RS; inner ramus lateral margin with spaced row of short RS, medial margin with sparse RS; outer ramus marginally shorter than inner.Uropod 2: peduncle with row of short setae; inner ramus nearly twice the length outer ramus, both margins sparse lined with RS; outer ramus, both margins with few short RS.Uropod 3: peduncle short, approx.0.3 × length of inner ramus, medial and inner margins of both rami with sparse row of short RS.Telson slightly longer than wide, u-shaped emargination 0.2 × lengths, lobes triangular, broadly rounded apically.Coleman (1994) based a detailed redescription of E. robusta on material from the Weddell Sea, while the type material of Epimeria robusta is from the Ross Sea.He found minute morphological diff erences between material of the opposing Antarctic shelves but interpreted them as intraspecifi c variation (Coleman 1994).Results of the phylogenetic analysis (see below) showed an Epimeria robusta species-complex comprising of a species each in the Weddell Sea and the Ross Sea.

Remarks
Th e new species, Epimeria robustoides sp.n. from the Weddell Sea (Figs 2-5; Fig. 10a) is morphologically very similar to Epimeria robusta Ross Sea (Figs 6-9, Fig. 10bd).Th ere is morphological variation amongst the E. robusta specimens from the Ross Sea: 1) in the relative length of pereonite 3 bearing a shallow mid dorsal keel and 2) coxae 2 and 3 are more acute in some specimen, not as obliquely truncate as in the pictured specimen.Th e morphological diff erences between Epimeria robusta and E. robustoides are summarized in the following:  Coleman (1994Coleman ( , 2007) ) pointed out some morphological variation between the type specimen from the Ross Sea and a redescription of material from the Weddell Sea and Elephant Island.We studied more than 30 E. robusta specimens from the Ross Sea and all agree with the type description (contrasting the Weddell Sea specimen): having a keel pleonite only well developed on segment 3, the posterior margin of pleonite 1 is drawn into a tooth and short teeth occur only on pleonite 3 and urosomite 1.
Th e main diff erence to the Coleman (1994) description is that our animal has a rostrum reaching the end of the second article of antenna 1, whereas Coleman's redescription shows a rostrum just reaching the end of the fi rst article of antenna 1. Th e morphological variation of Epimeria species, including the high variability of rostrum length of E. robusta related to size has been studied in detail by Lörz (2003).
Our current genetic analysis shows that Epimeria georgiana contains at least two species (see below).Epimeria georgiana is very similar to E. rimicarinata and E.inermis.Epimeria georgiana has the lateral face of coxa 4 sculptured, posterodistal and apical margins concave, the distal margin of coxa 4 is not curved around the ventral body side; and bases 5-7 notched posteromarginally, whereas in E. inermis coxa 4 is smooth, shield-like curved, with a somewhat convex ventral margin and a straight posterodistal margin, slightly curved under the ventral body side.Bases 5-7 are excavate, but not notched.E. rimicarinata has similarly shaped coxae as E. georgiana, however, the dorsal carinae are bilobed from lateral view, there are additional dorsolateral teeth on pereonites 5-7 and rounded humps on pleonites 1-3; basis 5 is not notched, basis 6 with a posteromarginal tooth, but this is directing posteriorly and not ventrally as in E. georgiana.We assume that specimen(s) that key out to E. georgiana belong to a species complex containing more than the two species shown by the present genetic separation.We are currently collating material of the diff erent morphotypes of E. georgiana at the moment, but presently have too little material to discriminate suffi ciently what minor morphological diff erences are non-variable features.Potentially, specimen from South Georgia, Bransfi eld Strait, Palmer Archipelago, South Shetland Islands and the eastern Weddell Sea shelf may be distinct species.

Phylogenetic analysis
Partial COI mtDNA sequences for 31 amphipod specimens were generated to examine the intraspecifi c and phylogenetic relationships in Southern Hemisphere Epimeriidae (EMBL Assession numbers FM955279-FM955309, Table 1).In addition 17 sequences of Antarctic Epimeriidae, Iphimediidae and Eusirus cf.perdentatus were downloaded from EMBL, the latter two taxa as outgroup sequences (Table 1).

COI analysis
In the fi nal analysis dataset comprised 47 sequences of 28 species.Th e total length of the partial COI mtDNA sequence was 496 characters of which 274 were variable and 254 were parsimony informative.Th e mean nucleotide composition is A=0.27604,C=0.24216, G=0.16383, T=0.31794.Th e amino acid translation with invertebrate mitochondrial code revealed no stop codons.A heuristic search found three most parsimonious trees when transitions and transversions are weighted equally (length 1599, CI 0.3333, RI 0.6815).Th e consensus maximum parsimony tree is shown in Fig. 1.Th e HKY85 maximum likelihood tree (data not shown) was similar in its topology except for changes in the position of clades while the species composition within the clades was retained.Th e bootstrap values performed for the Maximum Likelihood analysis are given after the bootstrap values performed for the Maximum Parsimony analysis.
Th e tree inferred from maximum parsimony analysis was rooted with Eusirus cf.perdentatus (Fig. 1).Th e Iphimediidae (jk=93, bs=82/89) and Epimeriidae (jk=83, bs=64/80) formed well-supported monophyletic clades.Within the Iphimediidae two of the three anaylsed genera, Gnathiphimedia and Iphimediella, showed para-phyly, while only Echiniphimedia appeared to be monophyletic.Within the Epimeriidae the two species from the New Zealand seamounts (E.bruuni and E. horsti) formed a supported sister group (jk=98, bs=91/98) to the Southern Ocean species group (jk=96, bs=87/78).Th e latter group split into two clades of lower support, one containing seven species from the shelves of the Weddell and Ross Seas, the other comprising nine nominal species from the shelves and slopes of the Antarctic Peninsula, Weddell Sea and Ross Sea.Th e two species identifi ed as Epimeriella before, E. macronyx and E. walkeri, form a well-supported group (jk=88, bs=67/71).Th e specimens examined from Epimeria georgiana showed paraphyly, forming two groups.One group consisted of the two specimens from the Antarctic Peninsula (AF452341 and AY061802), the other of two specimens from the Weddell Sea (FM955299, FM955305).Epimeria robustoides and E. robusta form a well-supported sister group to E. inermis.Within the well-supported species Epimeria robusta (jk=100, bs=100/100) four haplotypes were identifi ed.Th ree haplotypes were found in E. inermis from the Ross Sea area, where the specimen from the Balleny Islands (FM955282) formed a sister lineage to the Victoria Land specimens.
Pairwise sequence divergences between and within the genera and species of the Epimeriidae and Iphimediidae were highly variable (Table 2, 3).

Intergeneric distances
Th e maximum uncorrected distances between epimeriid and iphimediid genera varied from 23.99 to 35.2% (Table 2).Within the genera of the Iphimediidae uncorrected COI distances varied from 21 to 31.1%.As the Epimeriidae were represented by only one genus, no intergeneric distances could be analysed.

Interspecifi c distances
Interspecifi c uncorrected COI sequence distances in the Iphimediidae varied from 7.9% (Echiniphimedia scotti to E. hodgsoni) to 29.5% (Iphimediella cyclogena to I. georgei) Echiniphimedia scotti collected from the Ross Sea has a genetic distance of 7.9-8.5 to the Echiniphimedia species E. waegeli, E. hodgsoni and E. echinata from the Weddell Sea.Th e Weddell Sea species have interspecifi c distances of 9.9-10.5% amongst each other.

Intraspecifi c distances
Analysing the intraspecifi c diff erences the partial COI gene showed 0.0-1.2%sequence divergence within the seven E. robusta specimens from the Ross Sea and 0.2% sequence divergence within E. robustoides from the Weddell Sea.Sequence distances of 0.0-2.4% were found between the four E. inermis specimens, collected at four stations within the Ross Sea.Th e four E. rimicarinata specimens were collected from three diff erent stations of the Ross Sea and had intraspecifi c distances of less than 2.1%.Th e two specimens of Epimeria walkeri collected in the Weddell and Ross Seas showed 5.04% sequence divergence while within Epimeria georgiana the two specimens collected at the Antarctic Peninsula varied by ~15% from the two speciemens collected in the eastern Weddell Sea.

Taxonomic implications
In amphipod taxonomy it is common to have small morphological distances for separation between genera and even families (e.g.Coleman and Barnard 1991).Th e three families examined in this study, the Epimeriidae, Iphimediidae and Eusiridae, have only a few characters distinguishing them.Th erefore members of the Iphimediidae and Eusiridae were chosen as outgroups in the molecular part of this study.Iphimediidae diff er from Eusiridae in having at least one of coxae 1-4 being pointed (Barnard and Karaman 1991).Th e family Iphimediidae only diff ers from the Epimeriidae in having at least one pair of chelate gnathopods and in lacking the mandibular raker spines (Coleman and Barnard 1991).Within the Epimeriidae, Metepimeria is separated from Epimeria by bearing a 3-articulate maxilliped palp, vs the 4 articulate maxilliped palp of Epimeria.Th e only morphological separation of Epimeria and Epimeriella was the latter having a drawn out pars molaris without triturative surface and the lower lip bearing a wide hypopharyngeal gap.However, it was questionable whether this is a strong enough character for a valid generic distinction.Lörz and Brandt (2004) measured the variability in width of the hypopharyngal gap within selected epimeriid species and found no signifi cant diff erences between Epimeria macrodonta and Epimeriella truncata.Lörz and Brandt (2004) discussed that Epimeriella shows plesiomorphic characters of Antarctic Epimeria and indicated that the genus should therefore be synonymised with Epimeria.In their phylogeny based on 106 morphological characters, they analysed two Epimeriella spe-cies grouped within the Antarctic species of Epimeria and formed a clade with Epimeria annabellae (Lörz and Brandt 2004).Th e habitus of these three dorsally smooth species is similar (see e.g.Coleman 2007).Th e present molecular investigation shows the analysed species of Epimeriella, E. macronyx and E. walkeri, amongst the Antarctic Epimeria clade, supporting the former morphological studies by Lörz and Brandt (2004) of paraphyletic genera.Based on the here presented genetic data and a detailed morphological evaluation of the weak morphological separation criteria between Epimeriella and Epimeria we herewith synonymise Epimeriella with Epimeria.Th e topology of the molecular phylogeny showed the iphimediid genera Iphimediella and Gnathiphimedia to be paraphyletic taxa.Th e Iphimediidae, like the Epimeriidae, require more detailed morphological and molecular investigations to reveal their taxonomic characters.

Phylogeny of Southern Hemisphere Epimeria
Th e molecular phylogenetic analysis revealed the presence of a New Zealand seamount clade and an Antarctic clade of Epimeria.Epimeria horsti, collected from the New Zealand Ghoul and Gothic seamounts, is genetically closest to E. bruuni collected from the Young Hicks seamount, Hikurangi Plateau in New Zealand.Even though the New Zealand species have a genetic distance of over 20% from any Epimeria in the Southern Ocean, these two Epimeria species from rather close geographic localities also show a very large genetic distance, nearly 20% (Table 2).Th e New Zealand specimens show a strong monophyletic support (Fig. 1) whereas the support for the monophyly of Antarctic species is not so high in the likelihood analysis (78 bootstrap value), but the parsimony analysis shows higher values (bootstrap 87, jackknife 96).Without sequences of Epimeria outside of New Zealand and Antarctic waters it is not possible to determine the origin of the species based on this data.One likely scenario is that epimeriid amphipods "populated" New Zealand waters many million years ago or that several colonizations from the Ross Sea shelf to New Zealand shores have taken place.Another even more probable scenario is that epimeriids are Gondwanan and became isolated during sea-fl oor spreading in the Cretaceous.Our hypotheses are that all non-Antarctic epimeriids are monophyletic.Lörz and Brandt (2004) studied the phylogeny of Epimeria via morphological characters, with exception of E. loricata the species studied occurring beyond Antarctic waters form a well supported clade with the following synapomorphies: produced and pointed ventral angle of coxa 5; midventrally pointed coxa 4; lateral surface of coxa 5 bearing bump or tooth; merus of P5-P7 not produced.Based on their morphological characters, the two recently described and redescribed New Zealand species, E. horsti and E. bruuni, would be part of this non-Antarctic clade.
Th e specimens identifi ed as Epimeria georgiana show a genetic distance of ~15%, a distance value that proved to separate species within the Epimeriidae.Th erefore we take this high genetic diff erence as evidence for dealing with a Epimeria-georgiana species complex consisting of at least two diff erent species.According to Coleman (2007) E. georgiana occurs at South Georgia, in the Bransfi eld Strait, along the Palmer Archipelago (latter two both Western Antarctic Peninsula), the South Shetland Islands, and eastern shelf of the Weddell Sea.Coleman noted (pers.com.) that the fourth coxa of E. georgiana specimen from the Antarctic Peninsula is quite diff erent compared to the specimen from the Weddell Sea.Th e latter specimen resembled E. inermis, but show hooks at the basis of pereopods fi ve and six.Väinölä et al. (2001) included cytochrome oxidase sequences from an Epimeria georgiana specimen in their "Phylogeography of "glacial relict" Gammaracanthus from boreal lakes and the Caspian and White seas", but did not note the exact sampling location.Th e E. georgiana specimens in this study are from the Weddell Sea and the Antarctic Peninsula.We will separate the two cryptic species keyed out to Epimeria georgiana following the identifi cation key of Coleman (2007) in the near future.
Previous analysis of the partial COI gene showed 0.0-2.2%sequence divergence within eleven specimens of the E. schiaparelli from the Ross Sea (Lörz et al. 2007) forming a distinct group within Epimeria.Th is intraspecifi c divergence within E. schiaparelli is much less than this group's divergence from E. macrodonta (8.93-8.38%), the most closely related species.Divergences between other species were much larger (e.g.12.02% divergence for E. similis and E. macrodonta) further supporting the conspecifi city of all specimens identifi ed as E. schiaparelli, despite conspicuous variation in morphological characters as pointed out by Lörz et al. (2007).
It is remarkable that the interspecifi c variation of the iphimediid genus Echiniphimedia is smaller between the Ross Sea species E. scotti and any of the three Weddell Sea species than any distance of the Weddell Sea species to each other (Tab.2).A possibility is that the origin of the genus Echiniphimedia is in the Ross Sea and it has "populated" the Antarctic shelf several times.However, the Ross Sea shelf has been overrun by grounding ice sheets several times during the last glacial maxima.According to literature records, Echiniphimedia scotti, E. hodgsoni and E. echinata are accounted to have circum-Antarctic distributions with occurrences in the Ross Sea and at the Antarctic Peninsula (Coleman 2007).No records are known from a species of Echiniphimedia below 720 m, with the exception of Echiniphimedia hodgsoni (1120m).Unfortunately not enough material had been available of any of the three species from both geographic distant locations and none suitably fi xed for genetic studies.
Th e interspecifi c divergence of Epimeriidae and Iphimediidae from the Southern Ocean compared with the studied Gammaridae is low (Meyran et al. 1997, Hou et al. 2007) but similar to the divergences discovered in Hyalella (Witt et al. 2006).A low interspecifi c divergence indicates a relatively recent speciation.One reason for a successful recent speciation could be their variety in feeding patterns (Coleman 1989, Dauby et al. 2001, DeBroyer et al. 2001).Examination of the mandibles (mouthparts) of some species underscores their specialised food preferences.Gnathiphimedia mandibularis, which feeds on bryozoan colonies, has hammer-like mandibles (non-cutting) to crush the bryozoans' calcareous (calcium carbonate) exterior.Th e mandibles of Echiniphimedia hodgsoni, however, have sharp cutting edges for biting through tough sponge tissue (Coleman 1989).Dauby et al. (2001) have identifi ed eight diff erent feeding types among Antarctic amphipods, members of Epimeriidae and Iphimediidae are suspension feeders, deposit feeders, deposit feeders coupled with predation, opportunistic predators, micropredatory browsing, macropredation coupled with opportunistic necrophagy.
Another explanation for the recent speciation could be the variety in modes of mobility (Dauby et al. 2001).Epimeriidae and Iphimediidae show a great variation in their ability to move around, from sedentary (Epimeria georgiana, Epimeria rubrieques) to highly mobile (Epimera walkeri).Th eir degree of mobility is closely related to their food preferences, with the less mobile species more likely to be suspension-feeders and the more agile more inclined to be predators (Dauby et al. 2001).
Th e colour variation of these families might also add to their rate of speciation.Th ese specimens are predominantly red.Some species, such as Epimeria inermis, occur in several colours.Epimeria schiaparelli, comes in two diff erent patterns: striped and speckled, DNA analysis proved that both forms are the same species (Lörz et al. 2007).
Many specimens are covered with extravagantly long spines.We can only speculate on the role of these spines, since we know so little about the creatures' biology.A spiny exterior may off er protection from predators by breaking up the body outline and making the animal harder to see, or by rendering it unpleasant to eat.Echiniphimedia hodgsoni lives in sponges, and its many small white spines camoufl age it within the sponge tissue.
Th e diversity of microhabitats and of potential foods combined with the diff erent mobility patterns most likely encouraged the spread and speciation of Southern Ocean amphipods.
Th e two species are morphological very similar; the table above shows the morphological features separating Epimeria robusta and E. robustoides.Previous collections of Epimeria robusta from the Weddell Sea most likely have to be treated as fi ndings of Epimeria robustoides, unless further genetic studies reveal a sympatric distribution of E. robustoides and E. robusta.
Epimeria walkeri shows a genetic distance of 5.04% between the Ross Sea and the Weddell Sea.A COI sequence divergence value of >4% is often applied for separating marine invertebrate species in molecular barcoding (Witt et al. 2006).Since our data either show intraspecifi c variation of less than 2.5%, and an interspecifi c variation of at least 8.4%, we suggest that Epimeria walkeri is in the process of speciation.
Th e taxonomic relationships within the nominal Epimeria-georgiana-group were discussed above.Th e genetic sequence distances of ~15% between specimens from the Antarctic Peninsula and the eastern Weddell Sea are enough evidence for the existence of two species and to state that E. georgiana does not have a circum-Antarctic distribution but consists of a complex of cryptic species.
High values of intraspecifi c mitochondrial gene sequence divergence (COI and 16S mtDNA) indicating the existence of cryptic species are not only found in Antarctic species of Amphipoda from distant localities on the Southern Ocean.Similar results were found in studies on Isopoda (e.g.Held and Wägele 2005, Raupach and Wägele 2006, Raupach et al. 2007, Brökeland and Raupach 2008), Bivalvia (Linse et al. 2007), Octopoda (Allcock et al. 2004, Strugnell et al. 2008), Pycnogonida (Mahon et al. 2008), Crinoidea (Wilson et al. 2007) and benthic fi sh (Smith et al. 2008).Some reasons for the possible circum-Antarctic distribution of some breeding taxa is the dispersal via the Antarctic Circumpolar Current (ACC hypothesis) or extinction of a high proportion of taxa with pelagic development during vicariant events (extinction hypothesis) or the speciation enhanced in taxa with nonpelagic development in refuges during glacial maxima over the Antarctic Continental Shelf in the Pliocene/ Pleistocene (ACS hypothesis) (Pearse et al. 2009).Nominal species collected at several distant localities, for example from the eastern Weddell Sea and Antarctic Peninsula or the Weddell Sea and Ross Sea, resulted in the discoveries of species complexes.
Conclusions.Th e morphological and molecular analysis on the validity of the epimeriid genus Epimeriella Walker, 1906 confi rmed earlier the suggestion by Lörz and Brandt (2004) that this genus is a junior subjective synonym of Epimeria Costa, 1851.Five species are aff ected by this and are now named Epimeria macronyx comb. n., E. scabrosa comb. n, E. truncata comb. n., E. victoria comb. n. and E. walkeri comb. n.Th e analysed epimeriid specimens from New Zealand's seamounts and Antarctic localities formed two distinct clades separated by their geographic distributions.Within the Antarctic clade no further phylogeographic separation based on the species' distributions were observed.In order to evaluate the relationships between the Southern Hemisphere Epimeriidae, species from the Northern Hemisphere need to be included in the analysis.Th e use of the barcoding gene COI showed high sequence distances (12-13%) in the formerly circum-Antarctic distributed species Epimeria robusta and led to the description of Epimeria robustoides new species.Th e sequence distances within Epimeria georgiana of 15% between specimens from the Antarctic Peninsula and the eastern Weddell Sea gives evidence of another species complex in the Epimeriidae.Morphological variations in Antarctic amphipod populations from distant geographic localities have to be treated with care, potentially indicating the existence of cryptic species, all new to science.Based on our results, the hypothesis of circum-Antarctic species' distributions in brooding amphipods proved to be unlikely.

Figure 1 .
Figure 1.Maximum parsimony topology (length 1599, CI 0.33, RI 0,6815).Branch support values are given: jackknife above, parsimony / likelihood bootstrap below branches.Sequences obtained in this study are marked in bold.Th e scale bar gives the number of nucleotide substitutions per branch length.

Table 1 .
EMBL accession numbers, NIWA registration numbers and station data of specimens analysed.
Type species.Epimeria tricristata Costa, 1851 in Hope, 1851; by monotypy.Remarks.Th e most recent family diagnoses for the Epimeriidae is that of Coleman (

Table 2 .
Uncorrected genetic COI distances between epimeriid and iphimediid genera and species.

Table 3 .
Uncorrected genetic COI distances within selected Epimeria species