Landmark superimposition for taxonomic identification

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1 Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society The Linnean Society of London, 2004? Original Article LANDMARK SUPERIMPOSITION AND TAXONOMY J. M. BECERRA and A. G. VALDECASAS Biological Journal of the Linnean Society, 2004, 81, With 2 figures Landmark superimposition for taxonomic identification JOSE MARÍA BECERRA and ANTONIO G. VALDECASAS* Museo Nacional de Ciencias Naturales, C/José Gutierrez Abascal 2, Madrid, Spain Received 10 February 2003; accepted for publication 28 July 2003 The identification of organisms is a time-consuming task even for highly specialized researchers. Many ways to accelerate the identification process have been devised from pictorial keys to automatic, machine identification with different degrees of success. This paper explores landmark configurations as an aid to taxonomic identification. The basic hypothesis rests on the analogy between human fingerprints and organism landmark configuration. Translated into biological terms, it asks whether individual landmark configurations can be used as diagnostic characteristics for species identification. Water mites of the genus Torrenticola were used as test organisms. The results show that identification can be made simpler through the use of landmark configurations The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 81, ADDITIONAL KEYWORDS: geometric morphometrics Procrustes distances water mites. INTRODUCTION Biological taxonomy is the discipline of the description and identification of taxa (Vane-Wright, 2001). There has been some confusion surrounding the objectives of taxonomy, systematics and even morphology, but it is well understood that taxonomy offers a reference system for the biological organization of both living and extinct taxa (Heywood, 1988). How much biological information is implicit in this particular way of organizing living and fossil organisms is currently under discussion. At the same time, alternative ways of classifying are being proposed. Even the taxonomic procedures associated with the description of species, which underlie the Botanical and Zoological Codes, are being reviewed, especially given the ongoing discussion on the concept of species (Howard & Berlocher, 1998; Wilson, 1999; Wheeler & Meier, 2000). It is clear that taxonomists and geneticists refer to different things when they deal with species (rules that allow the investiture of a new species based on a single specimen, the type, do not fit well with disciplines whose main work is based on variance), although there is great concordance in the majority of cases. Nonetheless, taxonomy has provided a relatively robust reference system. *Corresponding author. valdeca@mncn.csic.es Both authors contributed equally to this study. The new approach to morphometrics (Rohlf & Marcus, 1993; Marcus et al., 1996) has provided a novel quantitative aspect to the study of form and size, and has led to the development of a powerful methodology for studying the morphological variation of populations. However, besides clarifying the status of a few problematic taxa, the impact this methodology may have on taxonomic procedures is not clear. Taxonomists will continue working with very small numbers of specimens sometimes only one since obtaining large numbers is expensive and performed to the detriment of collecting specimens of other species. More importantly, in many cases only one specimen will ever be available. These single specimens can be so different to anything known that they deserve description and definition as a separate species even if no further specimens are found. This occurs with some frequency in palaeontology, but is known even in modern-day botany and zoology. It is assumed that these single (or few) specimens are representative of the morphology of the species. Description and identification are difficult tasks that require a lot of time, and there is often a lack of funding and indeed of specialists for many groups of organisms. This underscores the urgent need to develop methods that might accelerate and facilitate this work (Godfray, 2002). This paper explores the use of morphometric methods of superimposition of forms (Rohlf & Slice, 1990; Bookstein, 1991; Rohlf, 1999) in 267

2 268 J. M. BECERRA and A. G. VALDECASAS the identification of taxa. Although complete identification is not always possible, at least a reduction in the number of steps and the time required for proper identification can be achieved. The hypothesis underlying this work is that general configurations of landmarks carry with them the imprint of the species. To use a human analogy, fingerprints are individual-specific configurations, as are face landmarks. Species identification by diagnostic characteristics and fingerprint identification by distribution of minutia are conceptually identical: both refer to invariant characteristics of an object a person in the case of fingerprints and a species in the case of diagnostic characteristics. Our research attempts to determine whether landmark configurations work as diagnostic characteristics. Morphometric characteristics show variation, but variation due to age, sex or geographical distribution does not preclude their use. Species descriptions are full of statements such as bigger than and twice the size of, but while fingerprints are invariant in concept they also present variations in practice. This is well treated by Jain & Pankanti (1999), who refer to the problems of establishing correspondence between two fingerprints. Apart from ontogenetic variation there are also variations due, for example, to elastic distortion of the skin. This paper explores the possibility of specific configurations that could behave as fingerprints for particular species. It should be possible to study the degree of fit between a certain specimen against library records of species configurations. In biological terms, this paper evaluates whether landmark configurations, as used in individuals, has diagnostic value in the identification of species. This is an empirical question: they may or not be of any use. The status of diagnostic characteristics is similar to that of colour in Popper s example of white swans: colour can be used as a diagnostic characteristic until the first black swan is found. Something similar could be said of landmark configurations. MATERIAL AND METHODS The biological material used included specimens of the water mite genus Torrenticola (Acari, Parasitengona). This is a group of aquatic mites with a worldwide distribution, found mainly in the benthos and the interstitial water of rivers and streams (Cook, 1974). More than 60 Torrenticola species have been found in Europe (Viets, 1978). Torrenticola species have a dorsal and ventral shield, and show easily distinguishable sexual dimorphism. Characters useful for the identification of the species of this genus include, among others, the proportion of mandible claw and palp segment sizes, the structure of the capitulum and size of the pharynx, and the morphology of the penis. However, this approach requires dissection and proper orientation of the body parts. Even when done in batches of several specimens at the same time, a substantial amount of time is required. Lundblad (1956) provides a detailed description of 19 Torrenticola species and subspecies from central and southern Europe. Eight of these were described for the first time by this author, who gave a uniform description of all of them, including standard body measurements and drawings of the dorsal and ventral surfaces, as well as the palps and other body structures. For our study, only the ventral surface of the mites was taken into account. Of all mite body parts, this carries the greatest number of landmarks. According to Bookstein (1991), landmarks gather together the whole set of possible measurements of a structure. Were used a total of 17 landmarks and pseudolandmarks, the definition and location of which are shown in Figure 1. Lundblad (1956) provides 32 drawings for the males and females of the species described; six drawings of males and females are missing. These drawings were digitized using a Canonvision EX1-Hi video camera connected to an OFG digital frame grabber (Imaging Technology) via a PC. This method provided two sets of type databases, one for the males and one for the females of each species. Due to the sexual dimorphism these species exhibit, analyses were performed separately on males and females. 9L 10SL 8L 7L 15SL 6L 5L 16SL 13SL Figure 1. Location of landmarks on the ventral shield of a specimen of Torrenticola. SL = pseudolandmark; L = landmark. 11L 14SL 4L 3L 17SL 2SL 12SL 1L

3 LANDMARK SUPERIMPOSITION AND TAXONOMY 269 Preliminary tests confirmed that the Lundblad landmark configurations were reliable, i.e. control configurations agreed with the specimens from which they came. To test this, the landmark configurations in Lundblad s drawings were digitized and compared against all the control specimens available. Each specimen was distinguishable from all others based on the selected landmarks. A set of 17 specimens (eight males and nine females of different species) from different areas of the Iberian Peninsula were then used in an identification test. These specimens were dissected with the help of a Zeiss Axiolab microscope, and schematic drawings of the ventral surface were made, including the location of the 17 landmarks and pseudolandmarks. The drawings were then scanned with an Epson Perfection 1200 Photo scanner. The images were not treated in any way. Taxonomic identification and identification by landmark configuration were performed independently by the two authors (A.G.V and J.M.B., respectively), working blind. All images were submitted to the process outlined in Figure 2. TAXONOMIC IDENTIFICATION The taxonomic identification of specimens was performed using the qualitative and morphometric data contained in the descriptions of Lundblad (1956) plus other data from the literature. None of these characteristics (Table 1) are directly connected with any of the 17 landmarks. MORPHOMETRIC IDENTIFICATION Coordinates for the 17 landmarks and pseudolandmarks (Bookstein, 1991) were obtained from the digitized drawings using TPS-DIG version 1.31 software (Rohlf, 2001) and stored as NTS files. Missing landmarks were digitized at the upper left corner of the image, and the coordinates then translated into a pair of predefined coordinates: -999, Each specimen s set of landmark coordinates were processed by the GRF-ND computer program (Slice, 1994), the specimen to be identified acting as the reference in the affine least squares (ALS) method (Rohlf & Slice, 1990; Rohlf, 1999). There are several superimposition algorithms. ALS was chosen for the simple reason that Drawing in paper scanner Drawing in image file Landmarks reference configuration image file TPS-DIG Unknown specimen scanner Male Type database TPS-DIG Landmark Coordinates Landmark Coordinates Lundblad (1956) Images of type drawings NTS format NTS format Female Type database GRF-ND List of type specimens ordered by Procrustes distance ORDER_PROCRUSTES batch or online analysis Sum of squared distances Affine least-square analysis batch or online analysis Figure 2. Identification process applied to an unknown specimen.

4 270 J. M. BECERRA and A. G. VALDECASAS Table 1. Main qualitative and morphometric characters used in taxonomic identification Dorsal shield: length and width Complete dorsal length Length, if free, of dorsal small platelets Lateral sides of dorsal shield: round or parallel Ventral shield: length and width Lateral view of capitulum: rostrum long/short/markedly curved Lateral side of capitulum with short or long extension Length of capitulum Length of chelicera Relation between claw and remain of chelicera Size of pharynx Size of palp segments Ratio P-II/P-IV palp Depth of capitular bay Distance between end of mandibular bay and beginning of genital field Genital field: length and width General look of genital appendix Position of excretion pore in relation to nearby glands: collinear, anterior, posterior it gave the best empirical results. In this sense, it has the same epistemological status that the UPGMA clustering algorithm has in phenetics (Sneath & Sokal, 1973). Since superimposition is rigid, the configuration in the data set that as a whole best fits the unknown specimen must be very similar to it. Thus, the ALS method removes differences in landmark configurations due to factors other than shape by scaling, translating and rotating the set of points in every known configuration to fit with the configuration being tested (the unknown specimen) (Slice, 1994). It also takes into account the affine transformation effects, i.e. the dilations (magnifications and contractions) for each specimen. The program produces a term Sd 2 : the lower its value, the better the specimens match. The results were saved as report files and processed by a program specially written in Delphi version 5.0 to obtain the Procrustes distances from Sd 2 (Rohlf, 1999). These distances were then organized in increasing order for all the specimens. The lower the Procrustes distance, the better the fit of a type specimen to the unknown specimen. The ALS method distributes all the differences over the entire configuration and removes uniform shape differences, increasing the likelihood of detecting more subtle local patterns (Rohlf & Slice, 1990). Procrustes distances (a global measure for each specimen in the type database ) provide a way of separating one specimen from any other. The residual error (i.e. the error due to the operator and the digitization system together) was obtained by taking the same configuration of landmarks from the same specimens ten times. COMPARISON OF METHODS After each author, working blind and using a different identification method, had reached decisions on the identity of the test specimens, the results were compared. RESULTS The results provided by the two identification methods are shown in Table 2. TAXONOMIC IDENTIFICATION The traditional characteristics for the identification of Torrenticola species include qualitative traits and measurements, such as the general body shape (with length/width measurements), and the size of the capitulum (details of the most important are in Table 1). More subtle variations noticeable to a familiarized eye include the lateral shape of the dorsal and ventral shields. Table 2 gives the identification of the specimens, including two that belonged to an undescribed species. Torrenticola (T) barsica (Szalay, 1933) (Specimens: males 1, 2, 5, 6; females 7, 8, 10, 11, 13, 14 and 15) The specimens described by Lundblad (1956) were sent to Szalay to check their identification by Lundblad himself. This species is close to T. elliptica although it has a different P-II/P-IV palp ratio. It is also similar to T. guadarramensis and T. hispanica. However, T. hispanica has a rounded body whereas T. barsica is longer. The anal pore is anterior in relation to the adjacent glandularia in the female of T. barsica, and posterior in T. guadarramensis. Furthermore, the sides of the dorsal shield are almost parallel in T. barsica and slightly rounded in T. guadarramensis. Lundblad based his description of T. guadarramensis on four specimens (one male and three females), but the species has never since been recorded. Torrenticola (T) brevirostris (Halbert, 1911) (Specimens: female 3; male 4) This is a roundish species with an anal pore colinear to the adjacent glandularia. It is easily distinguishable by its protruding pharynx. Torrenticola (M) madritensis (Viets, 1936) (Specimen: male 12) This species belongs to the subgenus Monatractides, and is characterized by the presence of hooks and protuberances at the end of Epimera I.

5 LANDMARK SUPERIMPOSITION AND TAXONOMY 271 Table 2. Identification obtained with morphometric and taxonomic methods Morphometric analysis Landmarks Pseudolandmarks Landmarks + Pseudolandmarks Taxonomic identification Sex A B C D1 D2 E F G1 G2 H I J1 J2 K L M ischnophallus T hispanica T ischnophallus T. barsica T ungeri T guadarramensis T hispanica T barsica T microphallus T barsica T madritensis M barsica T barsica T. barsica T barsica T hispanica T ischnophallus T ungeri T ischnophallus T ungeri T brevirostris T microphallus T microphallus T. brevirostris T hispanica T amplexa vs. T barsica T guadarramensis T hispanica T hispanica T madritensis M barsica T microphallus T. barsica T ungeri T hispanica T hispanica T stadleri vs. stenostoma M ischnophallus T barsica T madritensis M microphallus T hispanica T. barsica T ischnophallus T hispanica T ischnophallus T stadleri vs. stenostoma stadleri vs. stenostoma M barsica T barsica T. M madritensis M stadleri vs. stenostoma M. madritensis M amplexa T stadleri M madritensis M madritensis M brevirostris T brevirostris T guadarramensis T lusitanica M lusitanica M. New species hispanica T aberrata M aberrata M brevirostris T ungeri T brevirostris T brevirostris T lusitanica M aberrata M. New species aberrata M aberrata M lusitanica M hispanica T ungeri T stadleri M. F microphallus T ungeri T guadarramensis T. brevirostris T hispanica T brevirostris T hispanica T stadleri M aberrata M amplexa vs. tenuipalpis microphallus T amplexa vs. tenuipalpis T barsica T. barsica T hispanica T barsica T guadarramensis T barsica T amplexa vs. T microphallus T. T.

6 272 J. M. BECERRA and A. G. VALDECASAS Table 2. Continued Morphometric analysis Landmarks Pseudolandmarks Landmarks + Pseudolandmarks Taxonomic identification Sex A B C D1 D2 E F G1 G2 H I J1 J2 K L hispanica T amplexa vs. tenuipalpis T ungeri T. barsica T microphallus T amplexa vs. T amplexa vs. T amplexa vs. T ungeri T anomala T madritensis M amplexa vs. tenuipalpis T barsica T. barsica T barsica T amplexa vs. T guadarramensis T microphallus T barsica T hispanica T hispanica T brevirostris T hispanica T. barsica T microphallus T amplexa vs. T guadarramensis T guadarramensis T amplexa vs. tenuipalpis T amplexa vs. tenuipalpis microphallus T brevirostris T microphallus T. barsica T hispanica T microphallus T guadarramensis T guadarramensis T guadarramensis T ungeri T hispanica T hispanica T amplexa vs. T. barsica T brevirostris T anomala T amplexa vs. tenuipalpis amplexa vs. T amplexa vs. T hispanica T hispanica T amplexa vs. T guadarramensis T. barsica T ungeri T hispanica T amplexa vs. T guadarramensis T brevirostris T microphallus T hispanica T hispanica T microphallus T. barsica T guadarramensis T anomala T guadarramensis T ungeri T brevirostris T amplexa vs. T. T. T. A = unknown specimen #. Morphometric analysis: Using only landmarks: B = # of landmarks; C = Procrustes distances; D1 = species; D2 = subgenus. Using only pseudolandmarks: E = # of landmarks; F = Procrustes distances ; G 1 = species ; G2 = subgenus. Using landmarks + pseudolandmarks: H = # of landmarks; I = Procrustes distances; J 1 = species; J2 = subgenus. Taxonomic analysis: K = species; L = subgenus. M = male; F = female. T. = Torrenticola subgenus; M. = Monatractides subgenus.

7 LANDMARK SUPERIMPOSITION AND TAXONOMY 273 Torrenticola sp. (Specimens: males 16 and 17) These belong to the T. tenuirostris group of species (Viets, 1936) and will be described in detail elsewhere. They are characterized by the long curved front of the capitulum. No species of this group was included in the paper by Lundblad; they were used here to test morphometric identification. It is expected that the minimal distances of these specimens would be greater than that of all others in the database. MORPHOMETRIC IDENTIFICATION The mean residual error was d = ± Distances similar to or lower than this error were considered representative of identical or very similar specimens. Table 2 shows the values for the 17 specimens (eight males and nine females) used in this study. For each specimen, the first three most similar species are given in terms of landmark and pseudolandmark configurations, as well as both configurations together. Landmark analysis for males gave correct species identifications in four out of six possible cases. The two undescribed species should not be counted since, although the Procrustes method always tries to make a fit, there was no reference type for comparison. A poorer result was obtained with landmarks in females: there were only two out of nine correct identifications. However, if the doubtful species T. guadarramensis is considered to be T. barsica, a result of six out of nine is then obtained. For pseudolandmark identification, three out of six correct identifications were achieved for males (four out of nine if T. guadarramensis is included), and three out of nine for females (four out of nine if T. guadarramensis is included). Mixed analysis using landmarks and pseudolandmarks simultaneously gave five out of six correct identifications for males and two out of nine for females (six out of nine if T. guadarramensis is included). The Procrustes distances obtained for the two specimens of the yet undescribed species fell within the mid-range of the already known specimens. DISCUSSION The accurate identification of taxa is a very time-consuming business involving dissection and the treatment of the different body parts with products such as potassium hydroxide or other clearing agents. Identification is not an all or nothing endeavour: it tries to match new specimens with previously described organisms. If the fit is unsatisfactory something that a trained taxonomist should decide upon then the specimen deserves a description of its own. Obviously, everything has some similarity to every other living thing, no matter how far apart they are. A practical criterion in taxonomic identification is to accept diagnostic characteristics as providing sufficient evidence for similarity between a described species and unknown specimens. Morphometric identification by landmark configuration, however, gives continuous values from almost identical excluding error to any separation distance. In this study, the criterion used for morphometric identification by landmark configuration was to select the species which gave the lowest Procrustes distance in ALS superimposition. The ventral shield was selected because it has the largest number of landmarks and pseudolandmarks; other structures, such as the dorsal shield, have lower numbers of landmarks. The use of the palps, an articulated structure, also has its problems. The morphometric results indicate a certain degree of fit between the test specimens and the described target species, but not sufficient to allow an independent identification based on these alone. Single landmark configurations cannot be used as diagnostic characteristics with the same weight as are qualitative characters in morphology. A mixed strategy, using taxonomic and morphometric data might be a sensible way to deal with very similar species. Yet before this can be done a range of within-species variation in configurations is required if virtual morphometric types equivalent to taxonomic types are to be established. In morphometric identification, using more than one structure at once creates its own problems. In the case of a conflict in identification arising from the examination of two structures there is nothing more that can be done; there is no accepted criterion to decide between identifications arrived at from conflicting information. A detailed treatment of this problem is obviously needed. In the present case, it would seem that neither of the two identification methods can successfully be used alone. However, morphological identification includes measurable characteristics that could also involve morphometric procedures. Morphological identification has the benefit of relying on diagnostic characteristics that in many cases allow unambiguous identification. Morphometric methods give a degree of similarity and are not diagnostic in themselves (unless the presence/absence of a certain landmark is considered diagnostic). The morphometric method has the additional advantage that it uses public domain software which does not require a powerful computer, and a simple scanner can provide the images needed to digitize the landmarks. What seems clear, however, is that since morphometric identification always fits one specimen with another to some degree, the distance between them

8 274 J. M. BECERRA and A. G. VALDECASAS should not be used on its own as a criterion to define new species. Not explored in this paper, but potentially worth studying, is the use of geometrical morphometric techniques as identification tools for taxonomic levels above that of the species. To deal with these levels, especially genera and families, routines and criteria for handling missing landmarks should be available (Adams, Rohlf & Slice, 2004). ACKNOWLEDGEMENTS We thank Dr Ana Camacho for her useful comments and Jaime Rodriguez for the image in Figure 2. Adrian Burton corrected an English version of this paper. This work was partially financed by grant REN from the MCYT and REN GLO. REFERENCES Adams DC, Rohlf FJ, Slice DE Geometric morphometrics: ten years of progress following the revolution. Italian Journal of Zoology 71 in press. Bookstein FL Morphometric tools for landmark data. Geometry and biology. Cambridge: Cambridge University Press. Cook DR Water mite genera and subgenera. Memoirs of the American Entomological Institute. 21: Michigan: The American Entomological Institute, Godfray HCJ Challenges for taxonomy. Nature 417: Heywood VH The species concept as a socio-cultural phenomenon a source of the scientific dilemma. Theory Bioscience 117: Howard DJ, Berlocher SH, eds Endless forms. Species and speciation. New York: Oxford University Press. Jain A, Pankanti S Fingerprint classification and matching. Available at: pubs/sharat-handbook.pdf. Lundblad O Zur Ketnnis süd- und mitteleuropäischer Hydrachnellen. Arkiv För Zoologi 10: Marcus LF, Corti M, Loy A, Naylor GJP, Slice DE, eds Advances in morphometrics. NATO ASI Series, Series A: Life Sciences 284: Rohlf FJ Shape statistics: Procrustes superimposition and tangent spaces. Journal of Classification 16: Rohlf FJ TPS-DIG (Software for Acquiring Landmarks), Version Published by the authors. Available at Rohlf FJ, Marcus LF A revolution in morphometrics. Trends in Ecology and Evolution 8: Rohlf FJ, Slice D Extensions of the Procrustes method for the optimal superimposition of landmarks. Systematic Zoology 39: Slice D GRF-ND (Software for Generalized rotational fitting of n-dimensional landmark data). Revision Sneat PHA, Sokal RR Numerical taxonomy. San Francisco: W.H. Freeman. Vane-Wright RI Methods of taxonomy. In: Levin S, ed. Encyclopedia of biodiversity, Vol. 5. San Diego: Academic Press, Viets KO Wassermilben oder Hydracarina (Hydrachnellae und Halacaridae). In: Dahl F, ed. Jena: Tierwelt Deutschlands, 31 and 32: 10 & 574. Viets KO Hydracarina. In Illies J, ed. Limnofauna Europaea. Stuttgart: Gustav Fischer, Wheeler QD, Meier R, eds Species concepts and phylogenetic theory. A debate. New York: Columbia University Press. Wilson RA, ed Species. New interdisciplinary essays. Cambridge: MIT Press.