Hormogaster abbatissae Compilation of morphological and molecular data, a necessity for taxonomy: The case of Hormogaster abbatissae sp. n. (Annelida, Clitellata, Hormogastridae) Novo, Marta Fernandez, Rosa Marchan, Daniel Fernandez Monica Gutierrez, Cosin, Dario J. Diaz ZooKeys 2012 242 1 16 urn:lsid:zoobank.org:act:6A388AC5-A2E4-4A32-9BA4-F0F1C5684EBE Novo & Diaz Cosin Clitellata Hormogastridae Hormogaster CoL Animalia Hormogaster abbatissae Crassiclitellata 3 4 Annelida species abbatissae   Hormogaster abbatissaeNovo, 2010: 249 (eprints.ucm.es/12304/1/T32615.pdf) and Novo and Diaz Cosin, in press: (http://www.ucm.es/info/zoo/invertebrados/PDF/Novo%20et%20al%20%28en%20prensa%29%20When%20morphology%20and%20molecules%20clash.pdf) - nomina nuda superceded by current publication.  Material examined. Holotype. Adult (Catalog # SAN11 DZAF, UCM), 42°13'30.0"N, 2°14'57.5"E, from a small patch of forest near the Ter river, road C26, Km 210, between Ripoll and Sant Joan de les Abadesses, Girona (Spain), leg. M. Novo, D. Diaz Cosin, R. Fernandez, December 2006. Paratypes. 21 specimens (Catalog # SAN1-10, 12-22 DZAF, UCM), same collecting data as holotype. Other material examined. 16 Hormogasterspecies and several subspecies included in the study by Novo et al. (2011).  Morphological description. External morphology (Figure 1). Length of the mature specimens: 103-130 mm. Maximum diameter (pre-clitellar, clitellar, post-clitellar): 8, 11, 9 mm. Number of segments: 239-270. Weight (fixed specimens): 3.45-4.98 g. Colour: Anterior pink in live animals, with darker clitellum and grey-bluish posterior (Supplementary Figure S.1B). Specimens are grey-bluish when preserved in ethanol, with beige clitellum (Supplementary Figure S.1D). Prostomium proepilobic 1/3. Segments 1 and 2 showing longitudinal lines. Chaetae closely paired, quite lateral, visible along the body as two faint blue lines; intersetal ratio at segment 50, aa: 50, ab: 1.5, bc: 9, cd: 1, dd: 52. Nephridial pores in a row, between chaetae b and c. Spermathecal pores at intersegments 8/9, 9/10 and 10/11, at the level of chaetae cd. Male pores opening near the 15/16 as elongated fissures at the level of ab, showing heart-shaped porophores of variable developmental degree that can cover practically all width of the segment 15 and 1/2of 16 in mature specimens. Female pores in 14 more or less at the same level as the male ones. Clitellum saddle-shaped extending over 14,15-27. Tubercula pubertatis in (20) 21,22-26,27 appearing frequently as a continuous line in 21-27. Papillae with variable position, frequently situated at ab chaetae in segment 27, although more variable in other segments within the pre-clitellar and clitellar area. Internal anatomy. Funnel-shaped and strongly thickened septa in 7/8, 8/9 and 9/10, also in 6/7 and 10/11, less thickened though. Last pair of hearts in 11. Three globular strongly muscular gizzards in 6, 7 and 8 of shining appearance. Not apparent Morren'sglands, although in transverse sections of the oesophagus at segments 10 to 14 some thickened blood vessels can be detected, but never the lamellae typically showed by this glands. Lack of well-differentiated posterior gizzard, although the esophagus is a bit dilated at 15-16, but its wall is not especially muscular and its lumen does not exhibit a reinforcement similar to that in the anterior gizzards. In segments 17-25, 26, the gut showsfolds in the wall of every segment, forming what has been called a stomach in some earthworms. Typhlosole begins in 20, 21 and presents 15 lamellae, being the two lateral ones very small that therefore could be unnoticed. Number of lamellae gradually decreases, showing three from segment 80 to 140-150, and one until 160-170 where the typhlosole ends. Therefore the last 70 to 100 segments lack the typhlosole. Fraying testes and iridescent seminal funnels in 10 and 11. Two pairs of granular appearing seminal vesicles in 11 and 12 frequently showing black bodies. Ovaries and female funnels in 13; big ovarian receptacles in 14. Three pairs of spermathecae in segments 8, 9 and 10 included into septa 8/9, 9/10 and 10/11 the ones in 8 being the smallest. Spermathecae with the appearance of flattened sacks, dish or flying saucer showing irregular borders inside the body wall under some of the muscular fascicles. They can be divided internally into interconnected lobes that in fact do not represent independent spermathecae but simple multicameral spermathecae that open to the exterior by a unique pore. Anterior nephridial bladders V-shaped with widely open branches, being one of them shorter. They flatten towards the posterior section of the body, until the extent of showing appearance of an elongated sausage. In some of the specimens, the sexual chaetae in 11 and 12 present well developed follicles that go into the body as a projection where various chaetae simultaneously appear.  Distribution. Known only from its type locality.  Habitat. Specimens were collected in a small forest patch dominated by Populus alba, Acer pseudoplatanusand Rosa canina, which develops in a slope at the edge of a meadow. The soil was covered with abundant leaf litter (Supplementary Figure S1. A), and it is characterized by 13.57% of coarse sand, 9.62% fine sand, 6.27% coarse silt, 32.37% fine silt, and 38.18% clay, constituting a clay loam soil, carbon (C): 4.48%, nitrogen (N): 1.32%, C/N: 3.39, pH: 7.09.  Figure 1. External morphology of Hormogaster abbatissae. An illustration of nephridial bladders in segments 14 and 50 is shown in the upper right corner.  Etymology. The specific epithet derives from abbatissa, Latin for abbess, as the species is dedicated to the abbess Emma, the first Abbess head of the Monastery of Sant Joan de les Abadesses, founded in 885 AC by her father, the Count of Barcelona, Guifreel Pilos. The Monastery was run by nuns until the year 1,017 when the female community was expelled, presumably for disorderly conduct, and replaced by monks.  Molecular characters. Sequences from COI (8 individuals), 16S-tRNA (8 ind.), histone H3 (4 ind.), histone H4 (4 ind.), 28S rRNA (2 ind.) and 18S rRNA (1 ind.) were analysed with additional hormogastrid species. Phylogenetic analyses of the molecular data shows robust support for the monophyly of Hormogaster abbatissaesp. n., which is the sister species of Hormogaster sylvestrisQiu & Bouche, 1998 (Figure 2), described in the nearby locality of Montmajor (Barcelona, Spain). This clade forms the sister group to almost all other Hormogasterspecies from the NE Iberian Peninsula (see Novo et al. 2011for details). This latter clade from the NE Iberian Peninsula splits into two groups, the first clade including Hormogaster gallicaRota, 1994 from Banyuls-sur-Mer (S of France), Hormogaster catalaunensisQiu & Bouche, 1998 from El Brull (Barcelona, Spain) and Hormogaster pretiosa nigra Bouche, 1970 from Quillan (S of France). Its sister clade includes other Hormogasterspecies from the NE Iberian Peninsula, including Hormogaster riojanaQiu & Bouche, 1998 and related species (Figure 2). Uncorrected pairwise distances for 16S-tRNA and COI are shown in Table 3 for the sister species Hormogaster abbatissaesp. n. and Hormogaster sylvestrisand the morphologically-close Hormogaster riojanaas well as its sister species Hormogaster ireguanaQiu & Bouche, 1998. Hormogaster elisaeis included as a distant relative, even though it belongs to a possible new genus (see Novo et al. 2011). The networks recovered by Splitstree4 for the COI and 16S genes including morphological and molecular closest species are shown in Figure 2. GMYC analyses performed by Novo et al. (2012)identified Hormogaster abbatissae, Hormogaster riojanaand Hormogaster sylvestrisas different species.  Ecological characters. Soil characteristics in the localities where Hormogaster abbatissaesp. n., Hormogaster riojanaand Hormogaster sylvestrisoccur are shown in Table 4. Differences in soil textur ewere detected: Hormogaster sylvestrisand Hormogaster riojanainhabit Silt-loamy soils, whereas Hormogaster abbatissaesp. n. inhabits Clay-loamy soils. Hormogaster abbatissaesp. n. inhabits soils with a higher content in organic matter. Comparisons with the remaining species of the family were provided by Novo et al. (2012).  Figure 2. Top, part of the parsimony tree recovered by Novo et al. (2011), showing the clade where Hormogaster abbatissaewas placed (in that work it is named sp n.). Bottom, network representation for 16S-tRNA and COI recovered by SplitsTree4 of the closest species (surrounded by a black square in the tree above) and Hormogaster elisaeand Aporrectodea trapezoidesas distant references.The number of specimens used is indicated in parenthesis.  Table 3. Mean values of uncorrected pairwise differences in percentage obtained for 16S-tRNA (above the diagonal) and COI (below the diagonal, in bold) genes. Values of intraspecific differences are shown in the diagonal for the species that include more than one sequence type.    a eH. abbatiss  Hormogaster sylvestris  Hormogaster riojana  Hormogaster ireguana  Hormogaster elisae  aeH. abbatiss 0.10/   Hormogaster sylvestris 11.71 0.46/   Hormogaster riojana 17.80 17.36 0/   Hormogaster ireguana 16.11 18.58 9.53 0.33/   Hormogaster elisae 18.42 19.68 18.52 19.48 -  Table 4. Soil characteristics in the sampling localities of Hormogaster sylvestris(Montmajor MAJ), Hormogaster abbatissaesp. n. (San Joan de les Abadesses, SAN) and Hormogaster riojana(Alesanco, ALE). CSand: coarse sand, FSand: fine sand, TSand: total sand, CSilt: coarse silt, FSilt: fine silt, Tsilt: total silt, Tex: textural class, SL: Silt loam, CL: Clay loam, C: percentage of carbon, N: percentage of nitrogen, C/N carbon/nitrogen relationship.    CSand FSand TSand CSilt FSilt TSilt Clay Tex C N C/N pH  MAJ  SAN  ALE