Morphological distinction between estuarine polychaetes: Laeonereis culveri and L. nota (Phyllodocida: Nereididae)

The family Nereididae includes more than 500 polychaete species described worldwide, and includes species common in many benthic environments, but some other species may tolerate freshwater or can even thrive in humid substrates in tropical forests. In estuarine environments, nereidid polychaetes can be abundant and relevant as a food source for resident or migratory birds. Laeonereis culveri (Webster, 1879) is a common estuarine species found in tropical and subtropical Atlantic American shores and was described from New Jersey; its median and posterior parapodia have upper notopodial ligules usually longer than the lower ones, and the latter are parallel to the notaciculae throughout the body. L. culveri distribution is from Connecticut to central Argentina; however, this wide distribution might be due to the inclusion of several other species as junior synonyms, despite that some morphological differences were found between them. One of such species is L. nota (Treadwell, 1941), that was described from Texas; its parapodia have notopodial ligules of about the same size, and the lower ones are oblique to the notaciculae. In order to clarify the differences between these two species, and to define which inhabits the Northwestern Caribbean region, topotype materials from these two species and specimens from Chetumal Bay were collected, and their morphological features were compared. Our results indicated that L. culveri and L. nota are different species and that the latter is found in Chetumal Bay. On the basis of mature specimens, L. culveri is hereby restricted to the Northern Gulf of Mexico and Northwestern Atlantic Ocean, and L. nota is reinstated and its distribution extends from Texas, in the Gulf of Mexico to Chetumal Bay, in the Northwestern Caribbean Sea. A key to identify all species in Laeonereis Hartman (1945) is also included. Rev. Biol. Trop. 64 (1): 205-217. Epub 2016 March 01.

The polychaete family Nereididae de Blainville, 1818 includes species common in many benthic environments and has over 700 species in 45 genera worldwide (Read, 2015;Santos, Pleijel, Lana, & Rouse, 2005) Because they have been known since Linneaus and are frequently included in invertebrate zoology texts, this family is probably one of the best known among the polychaetes. Some nereidid species occur in estuarine or freshwater environments (Glasby & Timm, 2008), usually having more species than any other polychaete family in freshwater reservoirs (Foster, 1972); some species can even thrive in humid substrates in tropical forest trees (Glasby, Kitching, & Ryan, 1990;Glasby, Mogi, & Takahashi, 2003).
The morphological diagnostic features are well known for distinguishing genera among nereidids including prostomial, parapodial, chaetal and pharynx features, despite the fact that many undergo an impressive transformation of the body for reproduction (eyes become larger, and parapodia are transformed by a widening and enlargement of parapodial lobes and compound chaetal blades are replaced by wider, longer structures; a large part of their muscle bundles are reduced to release some room for gametes such that the body wall becomes transparent and delicate). This modification is known as epitoky and the morphological changes are so striking that after the first findings they were regarded and named as distinct genera. Among such taxa was Heteronereis Örsted, 1843 or Iphinereis Malmgren, 1866. It was through careful studies by Ehlers (1867) that they were shown as being reproductive stages of some already known genera. Reish (1957) indicated that epitoky is probably the most common method for reproduction among nereidids, but it is less common in estuarine or freshwater species (Herpin, 1925;Clark, 1961Clark, , 1977. The delineation of Laeonereis (Hartman, 1945), has been problematic and the progress on it deserves explanation. Hartman (1945) proposed this new name as a replacement name for the junior homonym to solve the homonymy between Leptonereis Kinberg, 1866 and Leptonereis Claparède, 1870. She selected Nereis culveri Webster, 1879 as the type species for Laeonereis and based the generic diagnosis on the notochaetae being homogomph spinigers, the neurochaetae including homogomph spinigers and heterogomph falcigers, and papillae occurring on both pharynx rings. Pettibone (1971:14) noticed that there were some problems with the diagnosis and emended it to indicate that notopodial spingers and neuropodial falcigers and spinigers were all homogomph, and that multifid papillae (actually verticillate bars) were restricted to the maxillary or distal ring, with two triangular papillae only on the oral ring (area VI). This generic diagnosis was followed by Hartmann-Schröder (1977:144), and Fauchald (1977:87).
Laeonereis includes estuarine species, often abundant and usually living in muddy substrates with high organic matter content (González-Escalante, 2001;Hedgpeth, 1950). Members of the genus are usually a significant food source in shrimp ponds (de Paiva & da Silva, 1998) or for migrating shore birds (Weber & Haig, 1997). In terms of karyotypic evolution, Laeonereis is also interesting because it has the largest number of chromosomes (2n = 38) (Ipucha, Santos, Lana, & Sbalqueiro, 2007) among the nereidids.
There is another problem regarding the species included in Laeonereis, which are only known from tropical and subtropical American estuaries. Hartman (1938Hartman ( :14, 1945 felt that L. culveri (Webster, 1879), originally described from New Jersey, also included L. acuta (Treadwell, 1923) described from São Paulo, Brazil. Pettibone (1971:14) concluded that there was a single species, L. culveri distributed from North Carolina to Uruguay and in El Salvador along the Pacific coast, and this concept was followed in a key to Grand Caribbean species (Salazar-Vallejo & Jiménez-Cueto, 1997). Pettibone (1971) followed Hartman (1938Hartman ( , 1945 and included as junior synonyms three other species: L. pandoensis (Monro, 1937) from a freshwater stream in Uruguay, L. nota (Treadwell, 1941) from an inner brackish water lagoon in Texas, and L. brunnea (Hartmann-Schröder, 1959) from estuaries in El Salvador. Further, Pettibone (1971) provided some illustrations for her specimens and showed some differences regarding the size and development of body features such as tentacular cirri, parapodia and pharynx.
Differences between supposed junior synonyms led previous authors to question such a large distribution. Orensanz and Gianuca (1974) concluded that there were morphological differences between L. culveri and L. acuta, and restricted their distribution with the former extending from North Carolina to the Gulf of Mexico, and the latter from Northern Brazil to the Valdés peninsula in Argentina. Pamplin, de Almeida andda Silva-Filho (2007) recorded L. acuta in Northern Brazil, andLiñero-Arana andDíaz-Díaz (2007) recorded it for the Southern Caribbean region. Likewise, L. brunnea was recently reinstated, and its distribution restricted to Pacific estuaries along Central America (Dean, 2001). A contrary perspective, based upon some morphological distortions due to preservation, led Oliveira, Santos, Lana and Camargo (2010) conclude that L. acuta is a junior synonym of L. culveri, but gave no details about a direct comparison of parapodial features.
Our objective was to assess which of the two North American Laeonereis species are present in Chetumal Bay by a comparison of their morphology and with topotype materials. The study of living specimens collected in Chetumal Bay, together with a comparison of preserved specimens from the same area and other specimens from Texas and North Carolina, revealed some interesting differences between them. In this contribution, important diagnostic differences are shown to distinguish two common estuarine species in tropical and subtopical Atlantic American shores: Laeonereis culveri (Webster, 1879), and L. nota (Treadwell, 1941), which is reinstated and can be found from Texas to the Northwestern Caribbean region, in estuarine areas. Topotype specimens are characterized and L. nota are reinstated. A key to identify all species in the genus is also included.

MATERIALS AND METHODS
Nereidid polychaetes were collected in Chetumal Bay, Quintana Roo, México, about 30 m from the shore at 1 m depth by shoveling fine sediment and sieving it through a 1 mm stainless steel mesh. Collecting trips were made, as detailed below, in April, 2011, July, 2012, September, 2013, and December 2014 Specimens were removed from the sieve with forceps and fixed in a 10 % formaline solution or 95 % ethanol. The formalin-fixed samples were later washed in tap water and preserved in 70 % ethanol. All specimens were deposited in the Colección de Referencia, ECOSUR. Nontype specimens from Texas and North Carolina were collected before and studied during research visits to the National Museum of Natural History, Smithsonian Institution, Washington (USNM), and the Los Angeles County Museum of Natural History (LACM). Field data information was copied from labels but no other collecting or processing information was available. Other specimens from the Mexican state of Tamaulipas, deposited in the collections of the Laboratorio de Biosistemática, Facultad de Ciencias Biológicas collections (UANL) were also studied. Pharyngeal features were not emphasized because of the condition of specimens which were often soft and fragile, and since there were important diagnostic details in parapodial features. A series of digital photographs were made with a Canon PowerShot G6 camera, a microscope adapter PerspectiveImage, and later plates were prepared by combining some of these photos with HeliconFocus (http://www.heliconsoft.com) or by hand.
Median and posterior parapodia modified ( Fig. 1D and Fig. 1E). Upper notopodial ligule digitate, as long as, or longer than, lower notopodial ligule; lower ligule blunt, subtriangular, about twice as wide as upper notopodial ligule, parallel to notacicula. Dorsal and ventral cirri blunt; dorsal cirri larger than ventrals, placed basally; basal glands decreasing in size posteriorly (rarely of the same size or larger posteriorly); ventral cirri displaced ventrally, difficult to observe.
Reproduction: Webster (1879:113) noticed that his specimens were in good condition after 48 h in fresh-water. Further, he (Webster, 1879:113) indicated that eyes "become a little larger … the body and feet are being swollen by the contained sexual products". No other modification of the relative size or shape of parapodial lobes was noticed. A better account, including parapodial ontogeny, dealing with apparently the same species was made by Mazurkiewicz (1975Mazurkiewicz ( , 2009) who studied a population in Mystic River Estuary, Connecticut. Specimens were found living in rust-colored, mucous-lined burrows; adults were brown whereas sexually mature specimens were green (Mazurkiewicz, 1975:187). Spawning occurs within burrows and larvae were present in the sediment flocculent layer, never in the water column (Mazurkiewicz, 1975:197-198). Apparently larvae progressed to the 3-chaetiger stage and then left the maternal burrow while spent females remained alive for 10-16 days after spawning. Larval chaetae were slightly bent and denticles were larger than in adults. As for parapodial development, Mazurkiewicz (2009: Fig. 3) showed that the upper ligule was markedly longer than the lower one in a 19-chaetiger larvae and, because this is a diagnostic feature, these observations are regarded as being based upon a Northern population of L. culveri.

Remarks:
Laeonereis culveri (Webster, 1879) differs from other species in the genus because it has elongated upper notopodial ligules in median and posterior chaetigers. This was clearly indicated in the original description (Webster, 1879) from New Jersey, and in other published accounts based upon North Carolinian materials (Gardiner, 1976:79).
Distribution: Originally described from Great Egg Harbor Bay (38°18'15" N -74°38'59" W), New Jersey, it has been found from Connecticut to Florida, and in the Gulf of Mexico, after Pettibone (1971: Fig. 7), it has recorded from Georgia, in estuarine environments especially in intertidal to shallow subtidal muddy substrates.
Median and posterior parapodia slightly modified ( Fig. 2D and Fig. 2E). Upper notopodial ligule triangular, blunt, shorter or about as long as lower notopodial ligule; lower ligule blunt, subtriangular, about twice as wide as upper notopodial ligule, oblique to notacicula. Dorsal and ventral cirri blunt; dorsal cirri larger than ventral ones, placed basally; basal glands decreasing in size and less pigmented posteriorly. Ventral cirri displaced ventrally, difficult to observe.
Neuropodial falcigers with blades 5-8 times longer than wide, finely denticulate, tip curved (Fig. 2F). Sperm plaques visible in median and posterior segments. Another specimen without posterior end had some sperm masses close to neuropodia in a few posterior chaetigers.
Reproduction: Klesch (1970) studied a population in Harbor Island, Redfish Bay, Texas and noted that immature adults were redorange, while sexually mature specimens were greenish, with adult males being light green to yellow and females deeper olive green. Mature specimens undergo widespread tissue histolysis including a great reduction of body wall musculature, septa and the intestinal tract. Klesch (1970:77) indicated that mature specimens spawn inside their burrows and that larvae were found within the burrows; spawned specimens usually die within the next 24 h. (Treadwell, 1941) resembles L. acuta (Treadwell, 1923) as shown in the key below, and both differ from L. culveri (Webster, 1879) since the latter has very long upper notopodial ligules in median and posterior chaetigers. There are several differences between L. nota and L. acuta, however. In L. nota the longest tentacular cirri reach chaetigers 2-3, and its dorsal cirri are half as Fig. 4. Laeonereis nota (Treadwell, 1941). Specimens temporarily stained with methyl-green. long as upper notopodial ligule, whereas in L. acuta the longest tentacular cirri reach chaetiger 5, and its dorsal cirri are one-third as long as upper notopodial ligule.

Remarks: Laeonereis nota
The specimens from Chetumal Bay are mostly soft bodied, probably collected after spawning, and this would explain their condition; however, because of their morphological features, they are regarded as conspecific with L. nota. The original description indicated that the holotype, and unique specimen, was blackish; however, no other topotype specimen shows dark pigmentation, and those from Chetumal are pale when alive as well. Specimens that are pale when preserved can take a dark pigmentation if they become partly dehydrated, if there was a black rubber sealer whose pigment often leaks into the fluid, or if China ink-labels were introduced before ink dried out into the jars. In some instances, this artificial pigmentation can be removed after surface brushing, and this could explain why we regard holotype pigmentation as a preservation artifact; further, no topotype specimen had any pigmentation remains.

DISCUSSION
The generic diagnostic features of nereidid polychaetes are rather conservative as there are usually two biarticulate palps, two antennae, usually four pairs of tentacular cirri and four eyes, and parapodia are usually biramous (Fauchald, 1977). There are a few genera with distinct modifications along the body such as branchiae (Dendronereis, Dendronereides), elytrae-shaped dorsal cirri (Kainonereis), or enlarged dorsal cirri in posterior parapodia (Alitta). Similar genera are usually separated upon some features of their pharynx armature or on the type of chaetae. Along the body of most adult, non-epitoke nereidids the parapodia change along one of two patterns, especially along median and posterior parapodia: they are either progressively smaller or become enlarged posteriorly. Laeonereis Hartman, 1945 is a well-delineated genus because its species do not undergo epitoky, have parapodia progressively smaller along the body, their pharynx has soft papillae and tufts of verticillate papillae, and compound homogomph falcigers in posterior parapodia. Pettibone (1971) disregarded the differences in parapodial features between the type species, L. culveri, and the other described ones. This was probably because it was known that nereidids undergoing epitoky show drastic transformations in their body, including parapodial features. However, it is now known that no species of Laeonereis displays epitoky and differences in parapodial morphology are here shown instead to be taxonomically significant, allowing species to be distinguished. Further, even eye size is not so markedly modified, and consequently the former inclusion of all species-name groups under the type species must be rejected. Mazurkiewicz (1975) found that early development occurs within Laeonereis burrows or tubes, and that their larvae live in the flocculent layer, not in the water column; this implies a low dispersal potential. If this type of larval development and behavior are widespread among Laeonereis species, they would have a very small dispersal potential, and their wide geographic distribution, even restricted to those ranges indicated above, would be difficult to explain. The answer may lie in migratory birds. Weber and Haig (1997) found that in two shorebird species (Charadriiformes), jaws of L. culveri were the main food item and that ingested worm size was related to birds' peak length. If this type of predation is size-dependent, probably birds could passively disperse smaller specimens or larvae within the sediment taken on feet and plumage. If there were only two species involved, they might seem too little for this distributional range, but there are more shorebird species in the region. For example, in Texas and close to the type locality of L. nota, Withers and Chapman (1993) counted 35 000 shorebirds of 26 species, and not surprisingly, the former author (Withers, 2002) found that they were especially abundant in Texas and Florida's SW coast in the United States, and in Laguna de Términos and Progreso, in the Yucatan Peninsula, Mexico. This type of dispersal is well documented among freshwater invertebrates (Bilton, Freeland, & Okamura, 2001;Frisch, Green, & Figuerola, 2007) in comparison to marine invertebrates, but because it seems to be also effective for those groups lacking dormant or diapause adaptations, it renders attractive for future research in marine environments. A recent paper by Simonis and Ellis (2014) has shown that bathing gulls can passively disperse rock-pool invertebrates, such that passive dispersal would include those birds not directly feeding on polychaetes as well.
Nevertheless, despite the fact that estuaries are very variable and stressful due to their daily and seasonal changes in salinity, temperature and organic matter content, there must be some restraints on cosmopolitan distribution of estuarine species. Distributional patterns must follow a set of limitations within the ecological horizon for each species (Salazar-Vallejo, Carrera-Parra, González, & Salazar-González, 2014) and defining distributional ranges or their limits is especially difficult in a progressively warming world, but worthy of further pursuit.

ACKNOWLEDGMENTS
Citlalli and Alejandro made summer research stays in Ecosur-Chetumal, and they wholeheartedly thank Emilia González-Vallejo for housing and feeding them. Funding for this research, including the visits to the museums of Los Angeles and in Washington was provided by ECOSUR. Sergio thanks the continued support by Leslie Harris and Dave Ocker (LACM), and Katie Ahlfeld, Geoff Keel, Linda Ward, Kristian Fauchald, and Karen Osborn (USNM). Cinthya Santos let us study some specimens of Laenoereis pandoensis from Brazil. J. Ángel de León-González and Víctor M. Conde-Vela read an earlier draft. However, the careful reading by four referees and by Daisy Arroyo resulted in a much improved document.