The diverse Grania fauna (Clitellata: Enchytraeidae) of the Esperance area, Western Australia, with descriptions of two new species

Seven species of the marine enchytraeid genus Grania Southern, 1913 are described from sediments sampled during the 2003 International Workshop on the Marine Flora and Fauna of Esperance Bay and the Recherche Archipelago, on the southern coast of Western Australia. Two species are new to science, the euryhaline Tasmanian G. dolichura Rota and Erséus, 2000 represents a new record for the state, and the remaining four species were known from other parts of Western Australia. Grania quaerens sp. n. is recognized by having a high chaetal index ( = short chaetal foot), small coelomocytes, penial apparati with long whip‐like terminal stylets, conspicuous spermathecae with ectally bulbous ducts, and ectally granulated ampullae housing sperm rings in their ental region. Grania sperantia sp. n. is readily distinguishable by the complete lack of lateral chaetae, a multiple‐banded pattern of the clitellum, extremely long sperm funnels, and the intrasegmental location of the spermathecal pores. The latter new species and four others in the collection (G. bykane Coates, 1990, G. crassiducta Coates, 1990, G. dolichura, and G. ersei Coates, 1990) are remarkable in possessing the head organ, a sensory structure unique to Grania that was not noted previously in Western Australian species. When considering the whole genus, the geographic pattern of the head organ appears southern‐centred: of the 17 species of Grania reported to possess it, as many as 13 inhabit the southern latitudes. The seventh species of the Esperance collection, G. vacivasa Coates and Stacey, 1993, is notable for the kind of items found in its gut and the unusual appearance of its pygidium.


Introduction
The town of Esperance is situated on the south coast of Western Australia, some 725 km east of Perth. Within its bay are around 100 small islands and 1500 islets and exposed reefs forming the Recherche Archipelago. The coastline is a rugged, high-energy coast, with spectacular white beaches between headlands dominated by dome-shaped granite hills of similar origin to the islands. The bay and the archipelago were named after two French frigates, L'Espérance and La Recherche, which sheltered in this area during heavy storms in 1792. The two ships were searching Australian waters for the lost expedition of La Pérouse, while charting the coastline and exploring the new continent.
The rich diversity of habitats and wildlife of the Esperance coastal area is well known and since the 1960s has been conserved by a number of national parks. Likewise, the islands, mostly inaccessible because of their steep sides and frequently extreme weather conditions, feature a variety of unique terrestrial environments and many notable plants, birds, reptiles, and mammals; since 1984 all but one are protected as nature reserves.
Not as much is known of the marine benthic flora and fauna of the area. To improve this knowledge, an international team of 25 marine biologists worked in Esperance Bay and the Recherche Archipelago for 17 days during February 2003, sampling and documenting the local biodiversity. The Esperance marine biology workshop was the seventh in a series organized by the Australian Marine Science Association, in conjunction with the Western Australian Museum and the University of Western Australia, to study marine biology in the western half of the continent.
In previous studies, 14 species of the marine enchytraeid genus Grania have been reported from Western Australia (Michaelsen 1907;Coates 1990;Coates and Stacey 1993;, making this state one of the best studied areas in the world with regard to this group of clitellate annelids. Nonetheless, knowledge about the presence of Grania along the southern coast is limited to the Albany region (Michaelsen 1907;Coates 1990). The next southern area of Australia for which data are available on Grania is Tasmania (Rota and Erséus 2000). The 2003 Esperance workshop provided an opportunity to obtain additional material from southern Western Australia, thus extending the geographical coverage of the continent's southern coast. Tubificidae, also collected at the Esperance workshop, were treated separately (Erséus and Wang 2005).

Material and methods
A range of intertidal and shallow subtidal sites spanning over 100 km in the Esperance area were visited by the second author (see Appendix 1). At each station, a sediment sample of a few litres was scooped by hand. Each sample was repeatedly (four times or more) stirred in seawater in a bucket, and the suspensions of organic material thus obtained were poured off into a 0.25 mm sieve, after which the sieved material was brought live into the laboratory for further examination. The Grania specimens were sorted out under a dissecting microscope. Additional specimens were sorted from subtidal sediment samples (also listed in Appendix 1) collected by various scuba divers. The worms were fixed in Bouin's fluid for 1 or 2 days and then transferred into 75% ethanol.
Later, the individuals were all stained in alcoholic paracarmine, dehydrated through an ethanol/xylene series and mounted whole in Canada balsam. All morphological studies reported herein were performed on these mounted specimens under a DM LB Leica microscope connected to a Coolpix 4500 Nikon digital camera. Drawings were made with the aid of a camera lucida. In the descriptions, specific segments are referred to by Roman numerals. Measurements were obtained by using an eyepiece micrometer. The total length of the chaetae was measured in a straight line from the distal tip to the furthest proximal point. The length of the ental hook (or foot) is meant as the projection of the maximal breadth of the chaeta at its ental end. The ''chaetal index'' , used to discriminate between different types of L-shaped chaetae, expresses the average ratio between the total chaetal length and the foot length (average calculated on n chaetae). The length of the glandular bulbs in the penial apparati was measured as their extension parallel to the longitudinal axis of the body. Only the main differential features of the head organ, where present, are given (for an overall description of this sensory structure see Rota and Erséus 1996;Rota et al. 1999).
Types and other reference material are deposited in the Western Australian Museum (WAM), Perth, in the Swedish Museum of Natural History (SMNH), Stockholm, and in the Museo Civico di Zoologia di Roma (MCZR), Italy.
Brain posteriorly indented. Head organ present, containing four equally large (ca 3 mm in diameter) globular inclusions ( Figure 1A) in both specimens. Pharyngeal glands with large ventral lobes (one pair) in IV in both specimens. Gut contents: few medium-sized diatoms.

Remarks
This Western Australian species, originally described from two stations in Princess Royal Harbour, Albany area (Coates 1990), is unmistakably recognized by the morphology of the spermathecae and penial stylets, and by the form and size distribution of the chaetae. The segment number is greater in the present specimens than in the types from Albany, but the holotype was admittedly incomplete (Coates 1990). In light of the new data, Grania bykane appears close to G. crassiducta Coates, 1990 (see below) as to chaetal shape, segment number and possession of head organ, but it clearly differs in morphometrics (having smaller body size, shorter sperm heads, smaller chaetae, higher chaetal index, shorter sperm and egg sacs), shape of penial stylets (with proximal constriction), chaetal size distribution, and other details. This is the first report of the occurrence of the head organ in G. bykane.

Distribution and habitat
Southern coasts of Western Australia (Albany and Esperance areas). Intertidal and subtidal to at least 6 m depth, in fine to coarse sand and in sediments under boulders and in seagrass beds (see Coates 1990).

Description
Body of the only complete specimen 7.15 mm long, 0.19 mm wide at V, 0.22 mm at clitellum, comprising 56 segments. Prostomium ogival, only slightly shorter than wide ( Figure 3B, C), 72-83 mm wide, 64-72 mm long; epidermis 4.5-8 mm thick at front, cavity of prostomium traversed by horizontal and oblique muscular laminae. Chaetae beginning in XVIII or XX laterally, in IV ventrally. Chaetae L-shaped, shaft straight, bluntly pointed ectally, gradually broadening toward ental end, until bending at 110u into a long, slender foot with indistinct heel, low instep, curved sole and flat toe ( Figures 2B, 3A). Preclitellar chaetae 55-80 mm long, with foot increasing in length from 13 to 27 mm; postclitellar chaetae 51-88 mm long, with foot decreasing in length posteriorly from 25 to 17 mm. Size of chaetae maximal in IX-XVIII (here, chaetal index 3.24, n53, s50.432), size gradually decreasing towards posterior body end. Somatic epidermal gland cells visible on segments I-II. Spermathecal pores as conspicuous oval buttons (orientated longitudinally) located in lateral lines, slightly posterior to 4/5. Brain posteriorly indented. Head organ present, containing four globular inclusions, each 3-5 mm in diameter ( Figure 3A, B). Pharyngeal glands with large ventral lobes (one pair) in IV in both specimens. Gut contents: brown organic matter, including some coarse fragments. Rectal ampulla not expanded. Nephridia not seen in front of clitellum.

Remarks
The morphology and morphometrics of the Esperance specimens of this taxon are in line with the original description from Princess Royal Harbour, Albany (Coates 1990). Conspecifics reported later from Rottnest Island (Coates and Stacey 1993), having shorter bodies, penial stylets almost twice as long as those of the types and a marked variation in width along the vas deferens, belong perhaps to an allied unnamed species. The occurrence of the head organ in G. crassiducta is noted here for the first time. The species appears now also remarkable for the low chaetal index (5 long ental foot), the bulbous shape of the spermathecal ducts (maximum diameter as great as or greater than maximum width of ampullae) and the thin horn-shaped penial stylets.

Distribution and habitat
Southern (Albany and Esperance) and western (Rottnest Island) coasts of Western Australia. Characteristic of lower intertidal to shallow subtidal habitats; in Esperance found in muddy coarse sand and in mixed sand with pebbles and coral.
Head organ containing two large (5-6 mm) inclusions in both specimens ( Figure 4B); other inclusions may be present but coelomocytes partly impede observation of the region. Anterior septa 7/8-8/9 thickened. Pharyngeal glands showing no ventral lobes in IV, nor any extra pairs in VII. Gut contents: brown organic matter and diatoms. Rectal ampulla spacious, bowl-shaped, filling most of pygidium, ending in a broad anus. Coelomocytes numerous, rather large (16-19 mm long), filled with small, non-transparent, round granules ( Figure 4A). Dorsal blood vessel originating in L. Sperm sac reaching XXV. Sperm funnels three to fours times longer than wide (130-170 6 38-45 mm), cylindrical, with tall hyaline portion below collar. Sperm heads 20 mm long, with evident helical structure. Vasa deferentia unmodified, showing conspicuous inner ciliation, 11 mm thick near funnel, then narrowing to about 6-8 mm thick. Penial apparati with bipartite aglandular sacs and trumpet-like, entally funnel-shaped stylets, latter 21-25 mm long, 7 mm wide at junctions with vasa deferentia, terminating in a 1.5 mm wide tip. Stylets fully developed in puberal specimen. Each stylet housed in anterior portion of aglandular sac. Egg sac not developed. Spermathecal ampullae bulb-or pear-shaped, with small but evident inner cavity, empty (both specimens precopulatory). Ectal ducts narrow (18 mm), ectally bent, slightly expanded but devoid of glands at orifice.

Remarks
Neither of the Esperance specimens of G. dolichura is fully mature but both are consistent with the original description (Rota and Erséus 2000) from Tasmania in diagnostic details of somatic (large size of head organ inclusions, long dorsal vessel, large chloragogen cells, thickening of anterior septa) and reproductive characters (clitellar pattern, penial bulb construction, stylet shape and size, length of sperm heads). From present observations on Tasmanian material (in E. R. collection), the two Australian populations are also consistent in the morphology of the rectum, a detail not noted previously. As concerns the pharyngeal glands, the Esperance specimens differ from the Tasmanian ones in lacking ventral lobes in IV and the extra glands in VII, while their sperm sacs extend more posteriorly. The new morphological information reported herein for G. bykane (head organ with four small globular inclusions, entally constricted penial stylets, etc.; see above) does not strengthen the possibility of a close relationship between the two species advanced by Rota and Erséus (2000).

Distribution and habitat
Originally reported from estuaries around Tasmania, where it is abundant and widespread (about 70 specimens from six stations) and covers a salinity range of 11-35psu, G. dolichura now appears to be a ''southern Australian element'' with a distribution possibly spanning across the Great Australian Bight and the South Australian Gulfs. Previously known as intertidal, in Esperance it is found in shallow subtidal (0.5-1.5 m) coarse sand colonized by Posidonia and in shelly heterogeneous sand. Coates, 1990 (Figures 4D-G, 5A) Grania ersei Coates, 1990 p 17-20, Figures 1A-D, 2;Coates and Stacey 1993, p 406-408, Figure 10A-F.

Remarks
The Esperance material gives the opportunity to complete the original and subsequent accounts of this Western Australian taxon (Albany : Coates 1990;Rottnest Island: Coates and Stacey 1993) with respect to the occurrence of coelomocytes, the different texture of the walls in the ectal and ental portions of the spermathecal ampulla, and the presence of a head organ (although not always detectable). The latter observation rectifies what was stated previously (Rota and Erséus 2000, p 249; based on material in C. E. collection). Apparently, there is considerable variation in chaetal size among populations from different geographic areas (Albany: 42-112 mm; Rottnest: 40-68 mm; Esperance: 50-75 mm), but the values of the chaetal index are likely to be typically low, due to the proportionally long ental foot (see Coates 1990, Figure 1B; Coates and Stacey 1993, Figure 10A, B). As noted in other species, the penial stylets (extremely long in G. ersei) appear fully developed before complete differentiation of the spermathecae has taken place.
South (Albany, Esperance) and west (Rottnest Island) coasts of Western Australia. In intertidal and subtidal sands down to 26 m (new depth record), often among boulders and pebbles, with algal debris. This species can be locally very abundant: both previous accounts were based on rich collections (Albany material: 112 specimens from 26 stations; Rottnest material: 101 specimens from 25 stations).

Etymology
The specific name is Latin for ''seeker, searcher'', in reference to its occurrence in the Recherche Archipelago.

Remarks
Diagnostic for this new species is the combination of a high chaetal index (5 short chaetal foot), small coelomocytes, long penial stylets forming loops inside the deep epidermal invaginations at male pores, conspicuous spermathecae with ectally bulbous ducts, and ectally granulated ampullae housing sperm rings in their ental region. The named features of the spermathecal ampullae (ectal granulation of walls and ental location of sperm rings), as well as the unusual length of the penial stylets, are reminiscent of the Western Australian G. longistyla Coates and Stacey, 1993 and G. ersei. In fact, at first sight one may have the impression that G. quaerens is but a small-sized version of G. ersei, with the shorter reproductive organs (spermathecal ducts and penial stylets are about one-half the length of those of G. ersei) being simply related to the smaller body size, but this is not so. The body size of G. ersei and G. quaerens largely overlap. Moreover, both G. ersei and G. longistyla have a different chaetal shape from G. quaerens (low chaetal index at least in G. ersei, see above) and lack preclitellar nephridia. Finally, as G. vacivasa, G. quaerens differs from G. ersei and the other species identified in the Esperance area by the lack of the head organ.

Distribution and habitat
South coast of Western Australia (Esperance). Exclusively found in lower intertidal, medium to coarse sand.  Figure 7A, B), 80-108 mm long, 133-175 mm wide at 0/1 (n520), epidermis just thinning at front (to 10-15 mm). Chaetae beginning in VII ventrally, absent throughout laterally. Chaetal length 125-163 mm in preclitellar segments, 105-160 mm in postclitellar segments, larger in (VII) IX-XXV (XXX), then gradually decreasing towards tail. Chaetae ( Figures 7F, 8A) stout, L-shaped, ectal tip blunt, shaft straight, progressively expanding entally (from up to 11 mm thick at mid-point to up to 14 mm across ankle), then curving into a short, slender foot with low instep and indistinct heel. Chaetal index 4.84, n510, s50.424. Sole of foot flat, tip of foot upturned. Epidermal gland cells inconspicuous. Thick layer of longitudinal body wall muscle fibres. Clitellum as much as 32.5 mm thick when fully developed, extending from the chaetae of XI to the whole of XIII, sometimes covering part of XIV ( Figure 7C, D); gland cells distributed into three main bands around body: a wider ''middle band'' of small (8-11 mm across), polygonal cells with two granulation types, hyaline and pale granular, interspersed in an irregular pattern-this middle band starts in the first half of XII; and an anterior and a posterior ''border band'', each of 14-16 regular transverse rows of cells, dominated by large rectangular (10 6 16-24 mm) hyaline cells. In closer view, the ''border bands'' indeed consist each of two sub-bands: one located at the (anterior or posterior) boundary of clitellum and comprising about 10-12 rows of merely hyaline cells, and the other, adjacent to the middle clitellar band, consisting of four to five rows of hyaline cells intermingled with few, very small (3-7 6 6 mm), refringent granular cells ( Figure 7D). Clitellum absent midventrally between male pores ( Figure 7C). Male pores ventrolateral in middle of XII. A small ''copulatory gland'' midventrally in XIV. Spermathecal pores ( Figure 8B) located in lateral lines at onethird of V. Brain indented posteriorly. Head organ housing four to six globular inclusions of uniform (4.8 mm) or different (2.5-6.4 mm) diameter ( Figure 7A, B); inclusions often showing a central hole (some specimens from ES03-28C and ES03-35A). Pharyngeal glands paired at 4/5-6/7, with ventral lobes in IV-VI; ventral lobes larger than dorsal ones in  IV. Gut contents: small to large diatoms, fragments of thick sponge spicules, organic material mostly decomposed. Rectal ampulla not expanded ( Figure 7G, H). First nephridia at 7/8; nephridia conspicuous in posterior body half. Coelomocytes not seen. Dorsal blood vessel commencing in XXXIX-XLIX (n512). Chloragogen cells reaching 16 mm above gut in anterior segments, more flattened (11 mm at most) behind clitellum. Sperm sac extending to XXII-half XXIX (n56). Sperm funnels elongate, 62.5 mm wide, about 19 times longer than wide, occupying 3.5 segments [XIII-XV(XVI)], with a short (16-19 mm) hyaline portion below collar. Heads of sperm reaching 22-24 mm above collars. Vasa deferentia not very long, always less deep than sperm funnels inside sperm sac (coils only seen as far back as XIV); vas unmodified, ciliated throughout, proximally 17.5 mm wide, ectally narrowing to 10 mm as approaching penial apparatus from posterior side. Penial apparatus, a compact glandular bulb, oval in lateral view, 96-112 mm long, slightly off-centred to the rear of the male pore, flanked laterally by an aglandular sac pointing posteriorly; all parts of apparatus anchored by robust muscles to body wall ( Figure 7E). No stylets present (penial type''3'' sensu Coates 1984). Egg sac extending into XXIX-XXXII (n54). Spermathecal ampullae large, oval, 88-112 mm wide, 104-128 mm long, with agranular walls, separately attached to oesophagus in posterior of V ( Figure 8B); about 30 sperm rings, maximally 19 mm wide, embedded in each ampulla. Ectal ducts 100-128 mm long, 32 mm wide at midcourse, narrowing at both ends; ducts S-shaped, proximally curved medially to enter ampullae laterally, distally bent at angle to reach external pores. Latter portion of ducts showing distinct muscle fibres.

Remarks
This new species is easily distinguished by the complete lack of lateral chaetae, the multiple-banded pattern of the clitellum, the extremely long sperm funnels, and the intrasegmental location of the spermathecal pores. Remarkable also is the high number (and the hollowness) of the inclusions that can be found in the head organ of some specimens. The lack of coelomocytes, consistently observed in material from four stations, could be an artefact due to loss of these cells on fixation (thus representing at least a behavioural, if not physiological, trait of the species). The body size, the chaetal characters (size, shape, and distribution) and the proportions of the sperm funnels are strongly reminiscent of G. conjuncta Coates and Stacey, 1993, another Western Australian species described (on a single specimen) from Rottnest Island, characterized by dorsally merging spermathecae and the lack of penial aglandular sacs. Grania sperantia, however, does not share such aberrant structure of the spermathecae and penial apparati, and is also readily distinguished from G. conjuncta by its regular number of pharyngeal glands, the occurrence of nephridia at 7/8, and a more posterior origin of the dorsal vessel.

Distribution and habitat
South coast of Western Australia (Esperance). In lower intertidal to shallow subtidal (2 m), medium to coarse sand. Coates and Stacey, 1993 (

Remarks
This large species, known so far from one station at Rottnest Island, is easily identified by the lack of lateral chaetae, the location of the largest chaetae at midbody, the midventral papillae in XIV and XV, the long sperm funnels (11-15 times longer than broad), and the unarmed penial apparati (Coates and Stacey 1993;personal observation). Three additional diagnostic features, noted herein for the first time and essential to clearly separating it from the otherwise similar G. sperantia, are: the high chaetal index (the highest value recorded so far in the genus), the lack of head organ, and the characteristic external and internal appearance of the pygidium. The dorsal blood vessel appears longer in the specimens from Esperance than in the types from Rottnest Island. The lack of coelomocytes, a feature not specified in the original description, could be due to a recurrent behavioural artefact (loss of these cells on fixation). Coates and Stacey (1997) noted a strong similarity between G. vacivasa and the northwestern Australian G. integra Coates and Stacey (1997), to the point that they suspected the two species might be identical other than in the presence of the penial stylet (in G. integra). Our own observations on the two species and present paper) confirm this discrepancy and add others: e.g. presence of abundant coelomocytes, confinement of the spermathecal ampullae to the anterior half of V, and no expansion of the rectal ampulla in G. integra. (The latter feature is possibly correlated with the kind of items found in the gut in G. vacivasa.) For the time being, however, the hypothesis of a close relationship between the two species should not be discarded.

Distribution and habitat
South (Esperance) and west (Rottnest Island) coasts of Western Australia. Subtidal to at least 11.5 m (new depth record), in medium to coarse sand.

Head organ
Five of the seven Grania species described herein from Esperance possess the head organ, a multicellular vesicular structure housing a number of solid inclusions, which is believed to act as a georeceptor in the peristomium (Rota et al. 1999). First discovered in five Antarctic and one deep-Atlantic Grania species (Rota and Erséus 1996), the head organ has subsequently been recorded in another five congeners scattered across the Atlantic, from the subantarctic South Georgia (Rota and Erséus 1997) up to Bermuda (Locke and Coates 1999), Bahamas (Rota et al. 1999), and the Canary Islands . Having been recognized elsewhere only in two Tasmanian species (Rota and Erséus 2000), the head organ was so far thought to be rare in species from the Indian Ocean and the Pacific. Our observations of its presence also in three known species of Western Australia (G. bykane, G. crassiducta, and G. ersei), one of which (G. ersei) had always been studied on abundant material, testify that this structure has been previously overlooked and could be widespread among the Australian fauna.
Today, the total of 60 species described worldwide in the genus are nearly equally distributed between the northern (30) and the southern (29) hemispheres; only the deepsea amphi-Atlantic G. atlantica crosses the equator, occurring between 48uN and 10uS (Coates and Erséus 1985;. Interestingly, the presently known geographic pattern of the head organ appears southern-centred: of the 17 species reported to possess the head organ, as many as 13 inhabit the southern latitudes. The relevance of this character to unravelling the evolution and biogeography of the genus has still to be investigated.

Biogeography
The Grania collection described above is comprised of only 70 specimens sampled at 20 stations in the Esperance area. The seven species differentiated in the samples testify to a high local diversity, even by comparison with earlier fruitful efforts exerted in Western Australia: about 125 Grania specimens belonging to six or seven species were yielded by sampling 33 stations during both the Albany and Rottnest Workshops (Coates 1990;Coates and Stacey 1993).
Of the recorded species, G. ersei was found at eight of the Esperance sites, spanning across the whole area explored, thus confirming it to be the most common species of Grania in Western Australia. This species and G. crassiducta live in all three marine areas surveyed in this state, whereas G. bykane is reported only from Albany and Esperance, and G. vacivasa is recorded from Rottnest and Esperance. None of these four species, however, is yet reported from outside Western Australia. Conversely, G. dolichura is known to occur all around Tasmania, thus representing one exception to the typically narrow geographical ranges noted in the genus. The dispersal capability of G. dolichura is possibly enhanced by its peculiar somatic traits and by its eurythermal and euryhaline nature (Rota and Erséus 2000). A similar case is represented in Europe by G. postclitellochaeta (Knö llner, 1935) (see . The observed species richness at Esperance and the occurrence of a Tasmanian species in southern Western Australia are not surprising. Esperance is located at the western border of the Great Australian Bight, and in biogeographical classifications of marine coastal areas of Australia (e.g. IMCRA 1996) it often marks the eastern limit of species proper to the South Western Province (a major region extending to Perth in the north) and the western limit of a suite of species reaching the Great Australian Bight from eastern regions, including the cold temperate Tasmanian Province. The combined presence of western and eastern elements can be attributed to the influence of the south-flowing Leeuwin Current that transports warm temperate fauna down the west coast and then eastwards into the Great Australian Bight, and vice versa the dispersal of eastern species in the opposite direction by the deeper Flinders Current, which flows from the southern tip of Tasmania to Cape Leeuwin (Cirano and Middleton 2004). Interestingly, a survey of the patterns of distribution of southern Australian echinoderms and decapods has revealed a minimal representation of narrow-range species near Esperance, whereas such species appear abundant near Perth, the South Australian Gulfs and at the southeastern tip of the continent (O'Hara and Poore 2000).
With the addition of the two new species described here, a total of 17 species of Grania have been recorded from Western Australian marine waters, representing 85% of all named Grania species reported from Australia, and 28% of all species described in the genus to date.