New light into the hormogastrid riddle: morphological and molecular description of Hormogaster joseantonioi sp. n. (Annelida, Clitellata, Hormogastridae)

Abstract The earthworm family Hormogastridae shows a remarkable disjunction in its distribution in the Iberian Peninsula, with the Hormogaster elisae species complex isolated from the rest of the species. Hormogaster joseantonioi sp. n., a new species found in the intermediate area between the main ranges (in Teruel, Aragón), was described following the integrative approach, as it is suitable for earthworms due to their highly homoplasic morphology. The phylogenetic analysis of the molecular markers placed the new species as a sister taxon to H. elisae, thus showing the colonizing lineage of Central Iberian Peninsula could have originated near the H. joseantonioi sp. n. current range. External morphological characters revealed some degree of overlap with previously described species, but internal characters presented configurations/states unknown from other members of the family. These traits make the new species a key piece to understand the evolution of Hormogastridae.


Introduction
The increasing availability of molecular and ecological data has placed the integrative taxonomy (as defined by Dayrat 2005) as a viable alternative to traditional species description. Several authors advocate its use in different animal groups (Padial and De La Riba 2010;Schlick-Steiner et al. 2010;Heethoff et al. 2011; but see Yeates et al. 2011 for iterative taxonomy instead) and particularly in earthworms Novo et al. 2012), whose taxonomy is in need of deep revision in the light of molecular phylogeny (Jamieson et al. 2002;Pop et al. 2003Pop et al. , 2007Chang et al. 2008;Briones et al. 2009;Pérez-Losada et al. 2009Novo et al. 2011;Fernández et al. 2012). Fernández et al. (2014) have developed a new tool based in micro-computed tomography to study specimens in a non-destructive way which could help as an additional source of information.
Taxonomic characters traditionally used for the study of earthworms are few and sometimes present high intraspecific variability (Michaelsen 1900 andStephenson 1930 on their global fauna; Pop et al. 2003 andBriones et al. 2009 about lumbricid earthworms). Recent findings show that cryptic diversity is common in these animals (but see critique in , therefore earthworm taxonomy can particularly benefit from an integrative approach. Novo et al. (2011) presented a molecular phylogeny of Hormogastridae (Oligochaeta, Annelida), whose taxonomy has historically been built on morphological characters, which highlighted some interesting evolutionary aspects. On one hand, hormogastrid distribution across the Western Mediterranean is biogeographically consistent, reflecting the geological events that affected the region in the Tertiary (which confirms previous studies, e.g Bouché 1972, Sbordoni et al. 1992. Two species -Xana omodeoi Diaz Cosin, Briones & Trigo, 1989 and the morphospecies Hormogaster elisae Álvarez, 1977 -, however, are found in locations far apart from the family main range in the Iberian Peninsula. While all the other Iberian species are distributed in Northeastern Spain, X. omodeoi inhabits Northwestern Spain and H. elisae is found in Central Spain (Segovia, Madrid and Guadalajara). The result is a disjunct distribution. Novo (2010) found Hormogaster elisae complex to be monophyletic, and thus the likely result of a single colonisation event presumably from the North or the East of the Iberian Peninsula. There could be remaining populations of the migrating lineage in the geographic gap, which haven't been discovered yet.
On the other hand, it seems that most key characters used for hormogastrid traditional taxonomy and phylogeny (notably the shape, number and position of the spermathecae) are highly homoplasic, showing little or no phylogenetic signal across the family.
Due to its relevance for this subject, the intermediate area between the main ranges of hormogastrids in Spain has been subject to recent sampling campaigns. Both Zaragoza and Teruel (Aragón, Spain) were suitable regions as they have been poorly sampled for earthworms unlike the surrounding provinces. While no success was met in Zaragoza, a population assignable to a new species of Hormogastridae was recently found in Teruel.
This paper focuses on the description of Hormogaster joseantonioi sp. n. from an integrative point of view, following the example of Novo et al. (2012). The new molecular and morphological data are interpreted to gain insight into the diversification and morphological radiation of the family, with some considerations about its constituent genera.

Earthworm specimens and sampling points
Specimens were collected by hand and fixed in the field in ca. 96% EtOH, with subsequent alcohol changes. Once in the laboratory, specimens were preserved at -20 °C.
Specimens available from previous studies (Novo et al. , 2011 of all known hormogastrid species were used for comparison. Morphological characters include those features traditionally used for hormogastrids and other earthworms.

Molecular data generation
Total genomic DNA was extracted from ventral integument tissue samples using the DNeasy Tissue Kit (QIAGEN) with two consecutive steps of elution (70 µl of buffer). Seven molecular regions were amplified: mitochondrial subunit 1 of cytochrome c oxidase (COI), 16S rRNA and tRNA Leu, Ala, and Ser (16S t-RNAs), one nuclear ribosomal gene (a fragment of 28S rRNA) and one nuclear protein-encoding gene (histone H3). Primer sequences, polymerase chain reactions (PCR) and sequencing reactions are the same as in Novo et al. (2011). GeneBank accession numbers for the holo-and paragenetypes, following Chakrabarty (2010) for the markers analysed here are shown in Table 1.

Phylogenetic analyses
The new sequences were combined with all the hormogastrid information available from previous studies (Novo et al. , 2011 in order to find their phylogenetic placement inside the family. Pontodrilus litoralis Grube, 1855, Dichogaster saliens Beddard, 1893, Amynthas robustus Perrier, 1872, Lumbricus terrestris Linnaeus, 1758 and Aporrectodea trapezoides Dugès, 1828 were used as outgroups (all the Gen-Bank accession numbers are shown in Appendix). As hormogastrid individuals from  (Darriba et al. 2012) was used to select the best-fit evolutionary model using the Akaike information criterion (AIC; Akaike 1973), and Bayesian information criterion (BIC; Schwarz 1978) which were GTR+I+G for COI, 16s and 28s, and HKY+I+G for H3.
Bayesian Inference (BI) of the phylogeny was estimated with MRBAYES v.3.1.2 (Ronquist and Huelsenbeck 2003) implemented in the CIPRES Science Gateway V. 3.3. (http://www.phylo.org/index.php/portal/). Unlinked nucleotide substitution models selected were specified for each gene fragment and the nucleotide substitution estimates were allowed to vary independently between each partition. Parameters were set to ten million generations and 10,000 trees were sampled for every 1000th generation, initiating the analysis from a random tree. After two analysis were performed 20% of the trees were discarded as burn-in, and the remaining trees were combined to find the maximum a posteriori probability estimate of phylogeny. Maximum likelihood analyses were performed with RAxML 7.2.7 (Stamatakis 2006) in the CIPRES Science Gateway with default settings, using GTR+I+G for each data partition and estimating the support for the resulting topologies by 100 bootstrap replicates.
Uncorrected pairwise differences for the mitochondrial regions were calculated between H. joseantonioi and the most closely related species with Arlequin 3.5 (Excoffier and Lischer 2010. To visualize the genetic distance we constructed networks with SplitsTree4 v.4.11.3 (Huson and Bryant 2006) for the more variable genes, including the former species plus Hormogaster riojana Qiu & Bouché, 1998 and A. trapezoides as outgroups. Default settings were used. Paratypes. Nine individuals (UCMLT 00001, 00002, 00004-00010), with the same collection data of the holotype.

Phylum
Other material examined. 16 hormogastrid species and several subspecies belonging to the UCMLT collection.
Morphological description. External morphology (Figure 2). *Measures taken on the two only complete specimens, one being the holotype.
Number of segments*: 305-369. Weight (fixed specimens)*: 7.05-11.57 g. Colour: From light brown to dark chocolate brown varying between individuals, with orangeish-brown clitellum of a lighter shade on living specimens (Figure 2a). Beige with brown stripes or patches, mainly on the anterior end, with darker clitellum on fixed specimens (Figure 2b).
Spermathecal pores at intersegments 9/10 and 10/11 at the level of cd. Male pores open over chaetae ab at the intersegment 15/16, surrounded by heartshaped porophores which extend over most of segment 15 and at least half of 16. Female pores in segment 14 at the same level as male pores.
Clitellum saddle-shaped extending over segments (13) 14-28. Tubercula pubertatis on 1/n 22-27(1/n 28) as a continuous line. Papillae of chaetae ab in variable positions, usually between segments 12 and 28: papillae on 12 always showing an unusual degree of development in mature individuals, being very conspicuous both in live and fixed specimens (Figure 2a).
Internal anatomy. Funnel shaped, strongly thickened septa in 6/7, 7/8 and 8/9, septum 9/10 slightly thickened. The latter's attachment to the dorsal body wall is displaced two segments backwards, creating a mismatch between inner and outer segmentation with an internally very wide segment 9.
Last pair of hearts in segment 11. Three shiny, strongly muscular gizzards in 6, 7 and 8. Not apparent Morren's glands, even though small wrinkles exist in the oesophageal wall between segments 10 and 16. A posterior gizzard is not well differentiated. There is a slight dilatation of the oesophagus between 14 and 16, but it lacks the muscular wall and reinforcements of a true gizzard. First section of the intestine is not dilated.
Typhlosole begins around segments 20 and 21 with seven lamellae, which around segments 26-27 increase to nine. From there they decrease gradually in number until segments 80-105, where they fuse in a single lamella. The latter extends until segments 218-230, where the typhlosole ends.
Fraying testes and iridescent seminal funnels in 10 and 11. Two pairs of voluminous, grainy seminal vesicles in 11 and 12. Ovaries and female funnels in 13, ovisacs in 14.
Two pairs of spermathecae in intersegments 9/10 and 10/11 (but apparently contained in segment 9 due to septum's backward displacement), the posterior pair bigger. They are sessile and disc-shaped, with multiple inner chambers which open to the exterior through a common pore, in the intersegments 9/10 and 10/11. Some individuals show double spermathecae (each multicameral and with own pore), either in 9/10 or 10/11 (Figure 3a).
Anterior nephridial bladders U-shaped with very close branches and no apparent cecum (Figure 3b). Bladders gradually flatten towards the end of the body, taking the usual elongated shape.
Distribution. Known only from its type locality.
The resulting Bayesian inference of the phylogenetic tree is shown in Figure 4. Its topology was congruent with that of the Maximum Likelihood inferred tree, except for the different placement of X. omodeoi. H. joseantonioi sp.n. was recovered as a monophyletic clade, with the H. elisae species complex as a sister clade.
Uncorrected pairwise distances for the genes COI and 16S-tRNA for H. joseantonioi and the species within the same clade (with H. elisae divided into its five cryptic species) are shown in Table 2.

Discussion
Both morphological and molecular characters of H. joseantonioi sp.n. separate it clearly from all known hormogastrid species, the number of typhosole lamellae and the kind and location of the spermathecae being particularly distinctive. Those characters, while failing to resolve internal relationships within Hormogastridae, have been shown to be suitable for species diagnosis (Rota, 1993  The species H. riojana, while distantly related according to molecular phylogeny, shows many similarities in morphology to H. joseantonioi (Table 3). However, H. joseantonioi differ by its lower number of lamellae in its typhlosole and shorter tubercula pubertatis. Moreover it is longer and heavier. While the two species share a very similar position and shape of the spermathecae, some H. joseantonioi individuals show an additional spermatheca in segment 10 (on the right or left side). These cases don't seem to be teratologic, as the supernumerary spermathecae have their own pore in the body surface and contain sperm, thus being fully functional.
Other hormogastrid species possess double or multiple spermathecae, but never of the multicameral, disc shaped kind.
H. joseantonioi sp. n. appears nested on a weakly supported clade on the phylogenetic tree, consisting in Hemigastrodrilus monicae, Xana omodeoi, Hormogaster pretiosa from Villamassargia, Hormogaster najaformis (and HPA from Omodeo, see Novo et al. 2011) and Hormogaster elisae. Genetic distances were high in all cases (16.47-19.08% for COI, 12.50-17.40% for 16S) according to the reference intervals given by Chang and James (2011). Aside from H. elisae, none of them showed significant morphological likeness to the new species, with the very different spermathecae configurations being noteworthy (Table 3). The H. elisae morphospecies was recovered as sister clade to H. joseantonioi sp. n. with high support. From a morphological point of view, most of their external characters overlap, except for a slightly longer clitellum and tubercula pubertatis, bigger average size and stronger pigmentation in H. joseantonioi sp. n. However, internal characters are very different and these species match neither in the number of lamellae in the typhlosole (five versus nine) nor in the structure of the spermathecae, which are tubular in H. elisae and disc-shaped and multicameral in H. joseantonioi. It's worth noting that H. elisae shares the backwardly displaced disposition of the 9/10 septum.
Based on their phylogenetic and morphological relatedness, an origin of H. elisae from a common ancestor with H. joseantonioi sp. n. seems likely. This scenario is sensible from a biogeographical point of view, as the locality of the new species is intermediate between the ranges of H. elisae and the northeastern main hormogastrid range. A connection of emerged lands would have been possible from the Cretaceous-Tertiary boundary onwards (Andeweg 2002).
While H. joseantonioi status as a good species and its phylogenetic relationships seem quite clear, generic assignment is a more problematic matter. Novo et al. (2011) recovered the genus Hormogaster as paraphyletic in their molecular phylogeny, pointing out the need for a deep taxonomical revision of the family Hormogastridae, currently in preparation (author's work in progress).