New light into the hormogastrid riddle: morphological and molecular description of Hormogaster joseantonioi sp. n. (Annelida, Clitellata, Hormogastridae) Author Fernandez Marchan, Daniel Author Fernandez, Rosa Author Novo, Marta Author Diaz Cosin, Dario J. text ZooKeys 2014 414 1 17 http://dx.doi.org/10.3897/zookeys.414.7665 journal article http://dx.doi.org/10.3897/zookeys.414.7665 1313-2970-414-1 8C6549A9B40D44478E3762224B9EAA17 8C6549A9B40D44478E3762224B9EAA17 Hormogaster joseantonioi Fernandez Marchan sp. n. Material examined. Holotype. Adult (UCMLT 00003), 41°0'55.68"N , 0°58'55.98"W , from a cleared holm-oak wood on the foothill of Oriche mountains, road A-2514 between Huesa del Comun and Rudilla, Teruel (Spain), collectors D. Fernandez Marchan and J.A. Fernandez Fernandez . Paratypes. Nine individuals (UCMLT 00001, 00002, 00004-00010), with the same collection data of the holotype. 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. Figure 2. (A) Live specimens of Hormogaster joseantonioi sp.n. External morphology of a fixed specimen, shown in a picture (B) and diagram (C). Length of mature specimens*: 178-180 mm. Maximum diameter (pre-clitellar, clitellar, post-clitellar) of mature specimens: 8-10, 9-11, 7-10 mm. 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). Prostomium prolobic, longitudinal striation on segments 1 and 2. Closely paired chaetae; interchaetal ratio at segment 40, aa: 33, ab: 1.3, bc: 6, cd: 1, dd: 27. Nephridial pores in a row between chaetae b and c (very close to b), visible on fixed specimens as a brownish line. 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 heart-shaped 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). Figure 3. A) Spermathecae in segments 9 and 10. Note the double spermathecae in segment 10 of this specimen. B) Nephridial bladder of segment 7. 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. Habitat. The specimens were collected at 10-20 cm deep in the soil in a cleared holm-oak wood, at the border between a dense forest of Quercus rotundifolia and a dryland farm. The soil had the following characteristics: 23.03% coarse sand, 8.06% fine sand, 5.33% coarse silt, 60.74% fine silt, and 2.84% clay, constituting a silty loam soil, carbon (C): 2.40%, nitrogen (N): 0.24%, C/N: 10.18, pH: 7.98. Mean annual temperature is 12.7 °C and mean annual precipitation is 447.2 mm, as indicated by the nearest weather station (in Herrera de Los Navarros, Zaragoza-23 km away http://www.aragon.es/DepartamentosOrganismosPublicos/Organismos/InstitutoAragonesEstadistica/AreasTematicas/14_Medio_Ambiente_Y_Energia/ci.05_Clima_Datos_climatologicos.detalleDepartamento?channelSelected=ea9fa856c66de310VgnVCM2000002f551bacRCRD#section1). Etymology . The species is named after Jose Antonio Fernandez Fernandez , father of the first author Daniel Fernandez Marchan and important contributor during the sampling campaign in which this species was discovered. Molecular characters. Analyses were conducted on sequences from loci COI (10 individuals), 16S (2 individuals), 28S (2 individuals) and H3 (2 individuals) of the new species, combined with similar sequences from other hormogastrid species. 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 Xana omodeoi . Hormogaster joseantonioi sp.n. was recovered as a monophyletic clade, with the Hormogaster elisae species complex as a sister clade. Figure 4. Bayesian inference of the phylogenetic tree on the concatenated sequence. Numbers above branches indicate posterior probability/bootstrap (of the Maximum Likelihood analysis) support values higher than 0.9/70 (shown as asterisks on terminal branches). Black rectangles show clades not recovered in both analyses (the alternative is shown with a dashed line). The cryptic species included in Hormogaster elisae are numbered from 1 to 5 (following Novo et al. 2010 ). Uncorrected pairwise distances for the genes COI and 16S-tRNA for Hormogaster joseantonioi and the species within the same clade (with Hormogaster elisae divided into its five cryptic species) are shown in Table 2. Table 2. Uncorrected pairwise distances for the genes COI (below the diagonal) and 16S-tRNA (above the diagonal) for Hormogaster joseantonioi and the species on the same clade. XAN - Xana omodeoi , HPRE - Hormogaster pretiosa , HNAJ - Hormogaster najaformis , HEM - two populations of Hemigastrodrilus monicae . Intraspecific divergence for COI/16S is shown in the diagonal.

HJOS	HE3	HE1	HE2	HE5	HE4	XAN	HPRE	HNAJ	HEM*	HEM**
HJOS