A new species of Heterospio (Annelida, Longosomatidae) from the Indian Ocean

Knowledge about the taxonomy and distribution of the monogeneric polychaete family Longosomatidae Hartman, 1944 is limited in all parts of the world. A new species of the genus Heterospio Ehlers, 1874 (Annelida, Longosomatidae) is described from the Indian Ocean. Heterospio indica sp. nov. is mainly characterised by the conspicuous flattening of the distal half of capillary chaetae located in anterior elongated segments. Several body characters of high taxonomic relevance in the genus are examined under the SEM and discussed. The 18S SSU rDNA and COI genes were sequenced and represent the first sequencing of a species of Heterospio, and the sequences have been deposited in GenBank.

The type species of the genus, Heterospio longissima Ehlers, 1874, was described from specimens collected off the coast of Ireland, NE Atlantic Ocean (Ehlers 1874).This species was subsequently reported in numerous localities in the same ocean and in the Sea of Japan (see fig. 8 in Parapar et al. 2014).In the Indian Ocean the only records of the genus correspond to one specimen of H. longissima off the coast of Sudan, Red Sea (Rosenfeldt 1989;Türkay 1996;Wehe & Fiege 2002), and two specimens off the Indian and Pakistani coast of the Arabian Sea (Hartman 1974;Wehe & Fiege 2002;Rao 2005; Kazmi & Naushaba 2013) (Fig. 1).
During a benthic investigation in shallow waters off the west coast of India, several specimens of the genus Heterospio were collected; they belong to a new species which is described herein, both morphologically (including SEM study) and genetically (including 18S SSU rDNA and COI sequences); information about its habitat is also provided.The distribution of the genus in nearby areas of the Indian Ocean is also reviewed.

Material and methods
This study is based on material collected by several sampling campaigns off the central west coast of India.Specimens were collected in different seasons across subtidal areas off Malvan, Ratnagiri, Mumbai and Veraval during 2013-2015 (Table 1).Samples were taken with a van Veen grab with a 0.04 m 2 sampling area.Most of the specimens were fixed in 5% formaldehyde in seawater solution, and then preserved in 70% ethanol for morphological identification.Some specimens were put directly into 96% ethanol for molecular studies.Samples were sorted at the CSIR-National Institute of Oceanography (CSIR-NIO), Regional Centre, Mumbai, India.All studied specimens were deposited in the collections of the Museo Nacional de Ciencias Naturales de Madrid, Spain (MNCN), Senckenberg Research Institute and Natural History Museum, Frankfurt (SMF) and CSIR-National Institute of Oceanography (CSIR-NIO), Regional Centre, Mumbai.
Observations, drawings and measurements of specimens were made with an Olympus BX51 compound microscope equipped with a drawing tube.Specimens were stained with methylene blue for examination of body and parapodia under the light microscope.For examination with scanning electron microscopy (SEM), specimens were dehydrated in a graded ethanol series, critical-point dried using CO 2 , mounted on aluminium stubs, covered with gold in a BAL-TEC SCD 004 evaporator, and examined and photographed under a JEOL JSM-6400 scanning electron microscope at the Servizos de Apoio á Investigación (SAI), University of A Coruña (UDC), Spain.
The description is based on the holotype; features of the posterior end of the body and chaetal fine structure (SEM micrographs) are from some paratypes.Intraspecific variation is also reported whenever recorded (e.g., segment size, branchiae arrangement).For the delimitation of body segments we follow Parapar et al. (2014); these authors propose that chaetae are located on the anterior border of segments and therefore the length of a segment should be considered as the distance from the chaetal bundle (or ring) to the chaetal bundle of the next chaetiger.For comparative purposes, one specimen collected by Rosenfeldt (1989) off the Sudanese coast in the Red Sea was also studied.
Molecular analysis was carried out at CSIR-NIO, Regional Centre, Mumbai.Genomic DNA was extracted with the DNeasy Blood & Tissue Kit (Qiagen), as per manufacturer protocol.18S SSU rDNA was amplified using primers 18SA and 584R resulting in 540-545 bp sequence-lengths.687 bp of COI was amplified with HCO2198 and LCO1490 primers (for primer references see Table 2).PCR mixtures for both genes contained 5 µl of Qiagen PCR buffer, 1 µl dNTPs, 38.8 µl ddH 2 O, 2 µl of each forward and reverse primer (10 mM), 1 µl template DNA and 0.2 µl Taq polymerase, making a total volume of 50 µl.Amplifications were carried under the following thermal conditions: 94ºC for 3.0 min, 30 cycles of 94ºC for 1 min, 48ºC (COI) or 50ºC (18S) for 1 min and 72ºC for 2 mins.A final elongation at 72ºC was carried out for 10 mins followed by cooling the reaction at 4ºC.Amplified products were tested on a 0.8% agarose gel.PCR products were purified with the PCR Purification kit (Qiagen) and sequenced bidirectionally by the ABi 3730XL Genetic Analyzer: 96 capillary sequencer.The final sequences have been deposited in Genbank.

Diagnosis (modified from Borowski 1994)
Body elongated, linear, divided in three regions.Anterior region with 6−8 short chaetigers; median region with greatly elongated segments; posterior region with few short, inflated segments.Posterior part of prostomium with a pair of lateral nuchal organs.Proboscis eversible, epithelial pouch.One pair of grooved peristomial palps, easily lost, leaving scars on lateral surfaces.Anterior region with 3-8 pairs of cirriform branchiae, usually very long, one pair per segment, arising above notopodia, beginning from chaetiger 2. Anterior region with biramous parapodia provided with simple capillaries, with or without acicular spines; elongated segments with chaetae usually forming a cincture near anterior margin; chaetae simple capillaries, may be accompanied by subuluncini, aristae chaetae, and acicular spines; posteriormost inflated chaetigers provided with strong acicular hooks.

Diagnosis
Eight anterior chaetigers short; chaetiger 9 (CH9) first elongated segment.Eight pairs of branchiae in fully developed individuals.Chaetae forming cinctures from CH10, provided with both thin and robust capillary chaetae arranged in two rows; both types of chaetae highly flattened in distal half; from CH14 robust capillary chaetae without fine distal end, subuluncini-like, thicker than on previous chaetigers.Aristate chaetae and acicular hooks not observed on elongated segments.

Etymology
The species is named after the Indian Ocean, where the specimens of the type series were collected.

Type material
Twenty two incomplete specimens and one posterior end were collected in 13 samples along the West coast of India (Table 1; Fig. 1).1).

Molecular identification
COI and 18S nucleotide sequences of Heterospio indica sp.nov were submitted to GenBank under the accession numbers: COI-KT259053, KU221229 and 18S-KT259051, KT259052.

Comparative material
One specimen collected by Rosenfeldt (1989) off the coast of Sudan, Red Sea (SMF 3795; labelled as H. longissima).
For a description of the posterior end, see below.
Fixed specimens creamy white in colour.

Variations
The first body segments show a certain degree of variation for two relevant taxonomic characters: relative size of first elongated segments and number/size of branchiae.The size of the first elongated segment (CH9) seems constant, about 2.5-3 longer than any short anterior segment (CH1-CH8); the relative size of following elongated segments is somewhat variable: CH10 is slightly shorter or longer than the anterior body region (i.e., from tip of prostomium to posterior end of CH9).This variability increases for CH11, which is longer than CH10 and may be longer than all preceding segments and prostomium together (cf.Fig. 6A vs 8A).Variability in sizes might be dependent on the degree of contraction of the specimen, but it may still be used as a taxonomic character (see Parapar et al. 2014); however, relative sizes of segments should be reviewed across the genus, because this character has not been properly assessed in descriptions of other species.On the contrary, variability in number of branchiae is likely to be size-dependent.Specimens show a wide range of number and sizes of branchiae that seem to be correlated with the size of individuals (i.e., width at level of anterior non-elongated segments; see Fig. 9).The presence and number of branchial pairs increases from anterior to posterior segments; thus, small specimens (<0.4 mm wide) bear four pairs while larger individuals (0.7-0.95 mm)  show up to 7-8 pairs.The presence and size of branchiophores and branchiostyles also shows certain variability, e.g., in the same specimen there are large and thin branchiostyles indistinctly on large and small branchiophores.
All specimens are broken at the level of the anterior elongated segments (CH12 to CH15) but a posterior region could be examined from a posterior end found in a Malvan sample in March 2015.Only one posterior region was found, composed of 7 elongated segments and a bulb-like inflated posterior end provided with 5 chaetigers, a terminal achaetigerous segment and pygidium (Fig. 3A).The last 5 chaetigers are provided with 2-4 acicular hooks (Fig. 3B); the anteriormost elongated chaetiger bears chaetae similar to those of CH10 of the holotype, with flattened distal ends; the chaetae of the other four elongated chaetigers are similar to those of CH12-14 of the holotype, i.e., of capillary and subuluncini type.

Specimen from Sudan
The specimen collected by Rosenfeldt (1989) off the coast of Sudan, which was identified as H. longissima (SMF 3795), has also been examined.It is in poor condition, i.e., incomplete, broken in two pieces, twisted and flattened dorsoventrally.According to its width (0.6 mm), this specimen fits within the range of large H. indica sp.nov.and it bears eight pairs of branchiae.Rosenfeldt (1989) highlighted the presence of palps which are now lacking.However, the relevant features of this specimen agree well with the description provided above of H. indica sp.nov.

Distribution and ecology
Heterospio indica sp.nov.was found off the west coast of India (Fig. 1) in shallow water (2.5 to 22 m depth) in mostly clayey silt and sandy silt sediments (Table 1).Hartman (1974) reported H. longissima based on two specimens from off the NW coast of India and Pakistan.In this article, Hartman (1974) only mentioned the papers by Ehlers (1895) and Hartman (1965) and did not provide any comparison with H. mediterranea or H. reducta, the two species described earlier by Laubier et al. (1972-73) from the Mediterranean Sea.Rosenfeldt (1989) reported H. longissima from Sudan (see above) based on a single specimen.She illustrated chaetae of the second elongated segment but did not provide any comparison with other species of Heterospio.Türkay (1996), Wehe & Fiege (2002), Rao (2005) and Kazmi & Naushaba (2013) mentioned these two records without any discussion of the identification of the reported material.We did not examine the material reported by Hartman (1974) but assume that it may belong to H. indica sp.nov.because of its geographic proximity to our material.The finding of the specimen originally reported as H. longissima by Rosenfeldt (1989) and identified here as H. indica sp.nov.(see above) extends the distribution of the new species to the Red Sea.

Key to world species of Heterospio
The key presented below is based on the provided by Bochert & Zettler (2009) but here the position and relative length of the first elongated chaetiger (FECH) and the number of pairs of branchiae have prevalence over chaetal types.The position of FECH in the species follows Parapar et al. (2014).

Discussion
The main taxonomic characters of the genus Heterospio are the number and length of anterior elongated segments, the presence or absence of palps and/or nuchal organs, the number of pairs of branchiae, and chaetal morphology (see Parapar et al. 2014).The study of specimens of H. indica sp.nov., however, allowed us to amend some comments given by Parapar et al. (2014).For instance, the presence of palps in Heterospio has been described and/or illustrated in several previous works (e.g., Hartman 1965;Wu & Chen 1966;Laubier et al. 1973;Borowski 1994); other authors, however, questioned their very presence (Uebelacker 1984;Bochert & Zettler 2009;Parapar et al. 2014) by interpreting the deep grooves behind the prostomium as nuchal organs and therefore not as palp scars.Examination of specimens of H. indica sp.nov.has clearly demonstrated that both palps and nuchal organs are indeed present.
As was previously proposed by Parapar et al. (2014), we consider that the number of short anterior segments and the relative length of the first elongated segments are two key characters in longosomatid taxonomy; nevertheless, they should be evaluated carefully because some variation was observed (see above).
Chaetal terminology in Longosomatidae is surprisingly variable given the small number of described species.The following terminology has been used for chaetae: aristate, capillary, stout capillary, subuluncini, transitional subuluncini capillary-like, spines and acicular hooks.A review of this nomenclature, in the frame of an eventual revision of the genus, seems necessary since some types might simply be transitional stages of the same type of chaetae, as has already been discussed by Borowski (1994).

Fig. 9 .
Fig. 9. Schematic drawings (not to scale) in dorsal view of some paratypes (MNCN and NIO) of Heterospio indica sp.nov., showing diversity of branchial arrangement in relation to size.Chaetigers numbered from CH1 (biramous) to CH10 (first flange-like).Width of anterior region indicated.

Table 1 .
Details of sampling locations off the west coast of India where specimens of Heterospio indica sp.nov.were found.

Table 2 .
List of PCR and sequencing primers.