GEOMETRIC MORPHOMETRIC ANALYSIS OF THE SHELL OF CERION MUMIA (PULMONATA: CERIONIDAE) AND RELATED SPECIES

Cerion mumia is a complex of eight subspecies distributed along the north coast of Cuba from Pinar del Rio to Camaguey provinces. The geometric morphometric analysis presented here was aimed at identifying patterns of shell shape variation to test the hypothesis of colonisation through land bridges during the Eocene-Oligocene. C. mumia cuspidatum, the easternmost population, was similar in shape to the subspecies from the north coast of Havana, but showed morphometric differences suggesting allopatric speciation followed by dispersal. The shells from the west were more globose than those from Havana or the east, which tended to be more cylindrical, as shown by the thin-plate spline analysis. As a result of the morphometric analysis I propose to elevate C. noriae comb. nov. and C. wrighti comb. nov. to species rank and to include C. noriae hondanum comb. nov. as a subspecies of C. noriae comb. nov. I report a second locality of C. noriae comb. nov. at Playa Santa Fe formation from the late upper Pleistocene. Geometric morphometric techniques are useful in species identification through comparing new samples with type material.


INTRODUCTION
The genus Cerion Röding, 1798 is endemic to the West Indies, with the distribution centre in Cuba and the Bahamas islands.The great geographic variation of its shell size, shape, sculpture and colour has made it a popular object of evolutionary research (Gould & woodruff 1986).The variation makes almost every population unique (Galler & Gould 1979).
Cerion mumia Bruguière, 1792 inhabits the northern coast of Cuba, including the provinces from Pinar del Rio, Havana, Matanzas in western Cuba and Camaguey in central Cuba, with several subspecies described based mainly on shell shape and colour (Pilsbry & Vanatta 1896, 1898, Pilsbry 1901, Clench & aGuayo 1953).
Some other species related to C. mumia share similar characters: cylindrical shells, usually quite high, coarsely ribbed, whitish or marbled and speckled, parietal lamella varying from rather long, with the inner end hardly visible in apertural view, to shorter or reduced, and rather convex whorls.Pilsbry (1901) included them all in the mumia group.
For many decades the taxonomy of the genus has been mainly based on conchological characters, resulting in virtually every population along the Cuban coast being described as a new species.Neighbouring populations are known to interbreed, suggesting that some of the nominal species reflect the geographic variation rather than the biological reality (Mayr & rosen 1956, Mayr & ashlocK 1969, Gould 1997).
Recent studies in Cuba used techniques of geometric morphometrics to find reliable evidence for the patterns of geographic variation (RodríGuezochoa 2014(RodríGuezochoa , 2015) ) and for a relationship between the shell shape and the geographical distance, supporting the patterns described by Gould & Paull (1977).Here I analyse the morphometrics of the mumia group and compare the material with the type specimens in terms of the classical variables of the shell to evaluate the relationships between the taxa in the current classification system.

MATERIAL AND METHODS
The species was sampled on the north coast of Cuba, after checking the bibliography (Fig. 1).Museum material from the following collections was examined: Smithsonian Institute, Field Museum of Natural History, Florida Museum of Natural History and Museum of Comparative Zoology, Harvard University who supplied valuable pictures of the types from their collections and the Cuban Ecology Institute (Table 1).In all, 207 shells were examined based on photos provided by the collections or on the samples collected by myself.Photos of all the collected shells were taken in situ with a SONY 12.0-megapixel camera, and the snails were released.
The shape description was based on the analysis of all the photographs using TPS 2.0 software which yields coordinated information from a total of 18 landmarks per sample (Fig. 2).The coordinates were subject to Procrustes superposition using PAST 3.0 software (HaMMer et al. 2009), to eliminate the effect of size differences and possible impact of rotation and translation at the time of taking the photograph or in the configuration of the landmarks.Consensus for each species was calculated using TPSRelw v.1.45(rohlf 2007) and subject to Principal Component Analysis (Jolliffe 2002) to obtain PC-scores for each species and prepare a geometric morphometric matrix.
All the multistate characters were entered in the geometric morphometric matrix which was then subject to Neighbor Joining analysis (saitou & nei 1987) with the Euclidean distance as the similarity index and with 5,000 bootstrap replicates.The material from the sampling localities was included in the matrix to check if the programme was useful for species identification and to prevent misidentification of the samples.In the phenogram Cerion alberti Clench et Aguayo, 1949 was used as outgroup.
Warp analysis was applied to the consensus, and thin-plate spline graphics was used to illustrate shape variation as deformation of configuration of landmarks on each consensus in relation to the mean shape.The warp scores were subject to principal component analysis (PCA) to evaluate the distribution of the taxa according to their shell shape (Michener & soKal 1957, cruz et al. 2012, Borda & raMirez 2014, RodríGuez-ochoa 2014).

RESULTS
In the phenogram the taxa form two well separated clusters (Fig. 15, for data see: Appendix 1), one with C. mumia noriae and C. mumia hondanum, the second including the remaining subspecies of C. mumia from Havana province and eastern Cuba.
I report a new locality of C. mumia noriae, Playa Santa Fe formation, in western Havana, extending its distribution range.The form was described as fossil from the sandstones of El Salado formation in eastern Havana (Clench & aGuayo 1953).Both geological formations date from the late upper Pleistocene.
In all the cases the museum material matches the sampling material showing that the programme was useful for species identification.
The thin-plate spline graphics (Fig. 16) shows that C. mumia chrysalis, C. mumia mumia, C. mumia fastigatum and C. mumia cuspidatum tend to have high  The PCA of the relative warp scores shows a geographical distributional pattern from east to west according to the shell shape (Fig. 17).

DISCUSSION
Cerion shows a great shell shape variation .The distribution of its shell shapes -random or presenting a pattern -has been a problem for decades (Gould et al. 1974, woodruff & Gould 1980, Gould 1997); some authors suggest that the actual number of species in Cuba is smaller than that of the morphotypes (Gould & Paull 1977, rodríGuezochoa 2014).
Our analysis using traditional morphometrics along with geometric morphometrics shows that C. mumia noriae should not be regarded as a subspecies of C. mumia.According to AGuayo & sánchez-roiG (1953) "This subspecies (C.mumia noriae) is the smallest of the mumia group and it is distinguished from the other known races, by not only its smaller size, but by the sharpened form of the axial ribs and by the dimensions of the shell, that are in general Our results suggest that the two taxa are not closely related.While C. mumia chrysalis is larger, with the characteristic radial irregular brown streaks and fine ribs (Fig. 18), C. mumia noriae has a small white shell with coarse sharpened ribs (Fig. 19).The geometric morphometric analysis revealed also differences in shape: the shell in C. mumia chrysalis is high, pointed and cylindrical with the apical expansion factor, while in C. mumia noriae it tends to be globose with the expansion factor on the last two whorls.Consequently, I propose to elevate the latter taxon to Cerion noriae comb.nov.
In the phenogram C. mumia hondanum is the closest to Cerion noriae comb.nov.They share the coarse sharpened ribs, small size, and the expansion factor on the aperture and the last two whorls, resulting in globose shells (Fig. 16).The last character is only shared by those two taxa and C. sanzi, but considering the particular globose shape of C. sanzi and its distribution in central Cuba, it seems likely that C. mumia hondanum could be a subspecies of Cerion noriae comb.nov.and not of C. mumia.Therefore I propose Cerion noriae hondanum as comb.nov.It differs from Cerion noriae comb.nov.mainly in its shell colour with roughly radial pale brown markings.
C. mumia wrighti occupies a basal position in the phenogram and resembles Cerion noriae comb.nov. in Fig. 18.Cerion mumia chrysalis, details of the shell showing the axial brown stripes pattern typical of this species, thinner ribs, and the presence of a small parietal tooth the pale shell colour, coarse ribs, and the shape with the expansion factor on the last whorl rather than Cerion noriae hondanum comb.nov.and Cerion noriae comb.nov., from which it differs in its larger size and more robust parietal tooth.Considering the differences compared to Cerion noriae comb.nov.and the results of morphometric analysis it can be concluded that C. mumia wrighti is not a subspecies of C. mumia; I propose to elevate it to species rank as Cerion wrighti comb.nov.It is noteworthy that all the subspecies of C. mumia share one morphometric character (Fig. 16): the apical expansion factor, resulting in higher, cylindrical shells.
According to the geographical pattern proposed by RodríGuez-ochoa (2014), species from eastern Cuba tend to have the apical expansion factor while those from western Cuba have the apertual expansion factor.In this study (Fig. 17), despite the limited number of taxa, the pattern is observable which conforms to the western pattern (Pinar del Rio province), with more cylindrical shells with an apical expansion at Havana and more to the east, suggesting a transitional morphospace which could support the allopatric pattern proposed by Woodruff & Gould (1980).The recently proposed zoogeographical history of the whole family Cerionidae based on molecular data supports the idea of colonisation from east to west through the Greater Antilles along the GAARlandia land bridge during the late Eocene to early Oligocene, and later to the Bahamas by stochastic accumulation of hurricane transported propagules (Iturralde-Vinent 2006, harasewych et al. 2015).
The use of geometric morphometrics in taxonomy and species identification has been well tested (Zelditch et al. 1995, rohlf 2002, SwidersKi et al. 2002, Conde-Padín et al. 2007, Cruz et al. 2012, Rattanawannee et al. 2012, Borda & raMirez 2014).In our case, the matching of samples with the type specimens proved to be a reliable method of species identification as long as types or reference material are available.
Relationships can also be inferred using morphometric shape variables (Rohlf 2002, toro et al.Here I used traditional morphometrics and geometric morphometrics to solve incongruences in the taxonomy of the genus Cerion.
The directional variation patterns of shell characters in Cerion indicate adaptive radiation in which ecological processes are as important as genetic ones.Further studies with the use of molecular data, which are essential for a thorough understanding of the evolution of this genus, will no doubt help to solve the taxonomy and systematic position of this large group in Cuba.

Fig. 17 .
Fig. 17.Plot of the results of principal component analysis (PCA) to the relative warp scores according to the distribution of the taxa along the Island of Cuba, showing distributional pattern according to the shell morphometrics

Fig. 19 .
Fig. 19.Cerion noriae comb.nov.details of the shell showing the white colour of the shell and coarse sharpened ribs.The dotted surface on the protoconch could result from erosion as this species is only known as fossil.The red colouration is typical of Playa Santa Fe formation deposits

Table 2 .
Morphological characters and taxa used in this study Fig. 15.Neighbour joining clustering of the Cerion species based on morphological distances, inferred from 5,000 replicates and generated by PAST 3.0 software (in red taxa from western Cuba, in green taxa from eastern Cuba and in blue taxa from Havana province)