Integrative Taxonomy, a New Approach to Answer the Questions in the Biosystematics: a Case Study of the Genus Gnopharmia (Geometridae)

A case study of the genus Gnopharmia 25

Entomologie heute 27 (2015): 25-35

Integrative Taxonomy, a New Approach to Answer the Questions in the Biosystematics: A Case Study of the Genus Gnopharmia (Geometridae)

Integrative Taxonomie, ein neuer Ansatz Fragen der Biosystematik zu beantworten: Eine Fallstudie an der Gattung Gnopharmia (Geometridae)

HOSSEIN RAJAEI

Summary: Looking back to the history of taxonomy reveals that thinking of the classiﬁ cation of organisms is an old engagement of human being. However, accumulation of knowledge, invention of new technologies and discovery of many biological facts and new conceptual deﬁ nitions open a new era of taxonomy. Currently, the classical taxonomy undergoes a transformation into an integrative taxonomy, which is a multidisciplinary approach for species delimitation. Thus, integrative taxonomy will be more trustworthy and, if worldwide established, faster in recognizing species and biodiversity. Using the genus Gnopharmia as an example, a geometrid moth that is distributed in the Middle East, Central Asia and Levant, the present survey shows the wealth of information (e.g. taxonomy, species boundaries, zoogeographic distribution and biology) which can be generated with an integrative approach.

Key words: Integrative taxonomy, morphology, geometric morphometry, phylogeography, DNA- barcoding, life history

Zusammenfassung: Die Geschichte der Taxonomie zeigt, dass Überlegungen zur Klassiﬁ kation von Organismen ein altes Anliegen der Menschen sind. Allerdings eröffnen die enorme Anhäufung von Wissen, die Entwicklung neuer Technologien und die Entdeckung neuer biologischer Phäno- mene sowie neue konzeptionelle Deﬁ nitionen eine neue Ära der Taxonomie. Zurzeit wird aus der klassischen Taxonomie eine integrative Taxononomie, die als ein multidisziplinärer Ansatz, u. a. zur Artabgrenzung, zu verstehen ist. Daher wird die integrative Taxonomie eindeutigere Ergebnisse liefern und, wenn erst einmal etabliert, auch schneller Arten und generell Biodiversität erfassen können. Am Beispiel der Gattung Gnopharmia (Geometridae), deren Arten ausschließlich in Mit- telost, Zentralasien und der Levante verbreitet sind, wird in der vorliegenden Übersicht aufgezeigt, welche Fülle von Informationen (z. B. Taxonomie, Artgrenzen, zoogeographische Verbreitungen und Biologie) mit Hilfe dieses Ansatzes zu erhalten ist.

Schlüsselwörter: Integrative Taxonomie, Morphologie, geometrische Morphometrie, Phylogeo- graphie, DNA-Barcoding, Biologie

1. Historical background velopment of the classifi cation of organisms (for details see JAHNS 2000). ARISTOTELES 1.1. From artifi cial to natural classifi ca- (384–322 B.C.), known as one of the most tion effective philosophers in ancient Greek, was one of the fi rst pioneers who thought As introduction into the present survey only on taxonomy. He formulated his thoughts some hints will given to characterize the de- on taxonomy and biodiversity in the book

Entomologie heute 27 (2015) 26 HOSSEIN RAFAEI

“Historia Animalium” (Greek: Περί Τά Ζώα fi nding the phylogenetic relationships was a ‘lστορίαι). ARISTOTELES had a hierarchical big dream and question. The question that view on the living organisms, which posi- needs around 100 years to be answered. tioned the low complex organisms in the Finally, the German entomologist WILLI lower level and those more complex ones HENNIG (1913–1976) overcame the problem in higher levels of the ladder of life (scala and developed a practical method for calcula- naturae). Furthermore, he was an essentialist tion of phylogenetic relationship based on believing that species are fi xed without any coded morphological data (see HENNIG 1950). changes in time. In this sense, he saw no HENNIG’s publication has transformed the intraspecifi c variation. So he tried to classify traditional classifi cation into “phylogenetic the organisms based on their similarities (e.g. systematics” (see JAHNS 2000). on their type of movement; he divided the animals into three groups: walkers, swim- 1.2. Species concept defi ne the ap- mers and fl yers). ARISTOTELES used a de- proaches of species delimitation scriptive binomial name for the organisms (e.g. he called human as featherless bipod). It seems that the process of species delimita- This attitude was subsisted until the 17th tion directly depends on the species concept, century, when several taxonomists started which the taxonomist are working with. new efforts. The typologists used the morphological In 1735, the Swedish naturalist CAROLUS LIN- similarities to classify the species, but after NAEUS (1707–1778) published his signifi cant DARWIN the evolutionists described species book “Systema Naturae” establishing the as lineages that evolve separately as own new taxonomy. Introduction of the bino- units (SIMPSON 1951). With his cladistic con- mial nomenclature and using a hierarchical cept HENNIG (1950) understands species as classifi cation system were two big steps unbranched lineages in a cladogram. Later, in upgrading the taxonomy. LINNAEUS and ERNST MAYR (1904–2005) summarized his the taxonomists, who followed him, also biological concept of species, which based belonged to the essentialism’s school using on reproductive isolation (MAYR 1970). morphological similarities for their classifi - Although the biological concept of species cation. However, LINNAEUS couldn’t explain encouraged the taxonomists to check the why the members of one taxonomic group reproductive isolation as a defi nite criterium (or taxon) show more similarity to each other for many species, the ecological concepts of than to the members of the other taxa. species (VAN VALEN 1976) draw attention to This very fundamental question was answered the environmental and ecological conditions over one century later by CHARLES DARWIN of a zone, which is occupied by a lineage. (1809–1882) in “The Origin of Species” Usage of these or other species concepts (1859). DARWIN argued that the similarities directly defines the methodological ap- of the members of one taxonomic group proach of taxonomy. Because each of these originate from their “common ancestor”. concepts has its own limitations, advantages The idea of a common ancestor led to the and disadvantages, an ultimate, standard development of a “natural” classifi cation procedure to answer all taxonomic questions system (vs. artifi cial system), in which the does not exist. members of a taxon share characters with common ancestors (phylogenetic signals). 1.3. Accelerating the taxonomy Today we have many algorithms, software and statistical methods for reconstructing Up to now, around two million species are the phylogenetic relationship, but for DARWIN described and it has been estimated that A case study of the genus Gnopharmia 27 between ten and 100 million eukaryote fi elds of Manihot (Manihot esculenta) in Africa species have not yet been discovered. Al- and its misidentifi cation is just one of many though the number of taxonomists strongly examples (SMITH et al. 2011). decreased, in the last 50 years the number Outbreak of the pest in the 1970s strongly of new described species was stable between decreased the yield of Manihot (as one of 14,000 and 25,000 species per year (PADIAL most important agriculture crops in Africa). et al. 2010). Taking a minimum quantity Besides using pesticides, a hymenopteran of undescribed eukaryote species (ca. ten parasitoid was imported from South Ameri- million), we need more than 400 years to ca to control this pest. However, none of describe nearly all biodiversity of the earth! these methods could effectively reduce the This is an important argument for thinking population of the mealybug. In the 1980s about acceleration of taxonomy and fi nding over 80 % of crop loss was reported. In novel methods for automation of species 1990 taxonomists realized that the pest identifi cation. species and therefore its related parasitoid Following this dream, development and were not correctly identifi ed. In the next implementation of DNA barcoding was step the right parasitoid (Anagyrus lopezi) a revolutionary achievement in taxonomy. was identifi ed and its introduction to the The idea of DNA barcoding seems to be fi elds successfully controlled the pest. The simple: fi nding a species-specifi c piece of economic loss due to a single taxonomic DNA (barcode); generating a library of this error was approx. over 20 million dollar piece of DNA for all known species; com- (SMITH et al. 2011). Such examples (which paring the barcode of an unknown species are not seldom) warn us not to sacrifi ce the with the known specimens and identifying accuracy of determination to the profi t of the species. an acceleration in taxonomy. As an optimal candidate, cytochrome oxidase I (COI) was realized as barcode (HEBERT 1.5. Integrative taxonomy: an alterna- et al. 2003) for many organisms, and during tive the last decade many taxonomists were convinced to use this method. However, Many taxonomists have recently acclaimed several new problems arose directly affecting the “integrative taxonomy” as a multidisci- the quality and accuracy of DNA barcod- plinary approach for species delimitation ing (e.g. introgression, co-amplifi cation of and for answering taxonomic questions nuclear copies of mitochondrial DNA, (PADIAL et al. 2010; SCHLICK-STEINER et al. heteroplasmy and contamination with dif- 2010). As highly complex systems, species ferent endosymbionts). Facing with these have multidimensional relationships with biological processes in a DNA barcoding their environment and other species. To survey may led to a completely different understand these systems we are forced not interpretation of the results and to a wrong only to check their appearance (morphologi- taxonomic decision. cal characters) or genetic data, but also their natural history, phylogenetic relationships, 1.4. Acceleration or accuracy? developmental stages, ecological demands, behavior and biology etc., in an objective There are many species misidentifi cations sense. In other words, when a complex his- in the taxonomic literature. Some of these torical background has infl uenced a popula- misidentifications cost much more than tion of organisms to have formed a unity one may imagine. The outbreak of Cassava (e.g. species), we may see this background mealybug (Phenacoccus manihoti) on agriculture in its genome, morph, behavior, niche

Entomologie heute 27 (2015) 28 HOSSEIN RAFAEI preference, etc. Looking at the species in an Geometric morphometry (GM): Land- integrative context gives us the opportunity mark-based geometric morphometry was to compare the validity of different concepts applied to the aedeagus of 298 specimens and regard the species valid, when different of Gnopharmia spp. to compare the results methods confi rm its unity. This approach of GM on species-specifi c characters with was used in many new taxonomic projects those of traditional morphology (see RAJAEI during recent years (including the genus et al. 2013a for more details). Gnopharmia Staudinger, 1892). Life history studies: Two Gnopharmia species were studied in their natural habitats 2. The taxonomic problems of the (RAJAEI 2010). genus Gnopharmia (Geometridae) Molecular taxonomy: Three nuclear se- resolved by integrative taxonomy quence regions (28S, ITS1 and ITS2) and fragments of one mitochondrial gene (CO1) The genus Gnopharmia Staudinger, 1892 is a were examined for their utility for phyloge- member of the family Geometridae (Geo- netic inference and species delimitation of meter moths), which is distributed only in Gnopharmia spp. (see RAJAEI et al. 2013a). the Middle East, Central Asia and Levant. Phylogeography: The genetic population Before the revision (RAJAEI et al. 2012), 19 indices of the Gnopharmia spp. were studied taxa (all species and subspecies) of this genus to explore a reasonable explanation for cur- were described by different authors (SCOBLE rent population distribution (see RAJAEI et & HAUSMANN 2007). High intraspecifi c varia- al. 2013a). tion in both genitalia and wing pattern led to Ecological survey: To estimate the current over 130 years misinterpretation of the spe- and past potential distribution dynamics of cies delimitations in this genus. Additionally, Gnopharmia spp. and their host-plants and to biology and food plants of Gnopharmoa spp. study the existence of any refugium in Iran were poorly known. Therefore, species identi- during the last glacial maximum (LGM) the fi cation and defi nition of the geographic dis- species distribution modeling was used (see tribution proved to be extremely diffi cult. To RAJAEI et al. 2013b for details). solve this problem, an integrative approach using a wide spectrum of methods was used 2.2. Results to appraise the taxonomy, distribution, phylogeography and biology of this genus. As a result of the morphological surveys, a series of new morphological diagnos- 2.1. Methodological aspects tic characters were discovered, e.g. tibial spines, octavals of the male pregenitalia To realize the integrative approach, over and arrangement of subapical spines on 2,000 specimens including the type series the aedeagus (see Fig. 1). Comparisons of of all species were studied. Specimens were all morphological characters revealed that collected or loaned from 14 museums and many synonymies could be recognized many private collections in Iran and various and the total number of valid taxa in this European countries. A variety of methods genus was reduced from 21 to ten taxa was used: (seven species and three subspecies). Two Morphological studies: Light microscopy taxa were transferred to the closely related and scanning electron microscopes were genus Neognopharmia Wehrli, 1953. One used to study the external and internal taxon (G. colchidaria cocandaria) is revived as anatomy of specimens (including over 900 valid species. One species (G. sinesefi da) was genitalized species; see RAJAEI et al. 2012). downgraded to a subspecies of G. colchidaria A case study of the genus Gnopharmia 29

Fig. 1: Gnopharmia rubraria. A Female genitalia. B Male genitalia. Scanning electron microscopy shows clearly the differences of the arrangements the spines on the subapical part of the aedeagus of (C) Gnopharmia kasrunensis, and (D) Gnopharmia colchidaria. Abbreviations: ad. = aedeagus, ap.ad. = apical part of aedeagus, b.c. = bursa copulatrix, p.a. = papilla analis, sa. = saccus, un. = uncus, v. = valva (see RAJAEI et al. 2012). Abb. 1: Gnopharmia rubraria. A Genitalpräparat eines Weibchens. B Genitalpräparat eines Männ- chens. Die rasterelektronenmikroskopischen Aufnahmen zeigen den deutlichen Unterschied in der Anordnung der Dornen auf dem subapicalen Aedeagus von (C) Gnopharmia kasrunensis und (D) Gnopharmia colchidaria. Abkürzungen: ad. = Aedeagus, ap.ad. = apicaler Teil des Aedeagus, b.c. = Bursa copulatrix, p.a. = Papilla analis, sa. = Saccus, un. = Uncus, v. = Valva (s. RAJAEI et al. 2012).

Lederer, 1870. Additionally, fi ve new records shows a very low genetic distance between of the valid taxa are reported for the fauna them (Fig. 3). Therefore, it was concluded of different countries in the Middle East that these populations belong to the same (see RAJAEI et al. 2012 for details). species and that the dark colored specimens Using only the data of descriptive mor- from Southwest Turkey are a melanistic phology didn’t help us in some cases to form of G. rubraria. Based on these new postulate a reasonable result. For example, results, the geographic pattern of all valid populations of Gnopharmia rubraria from species was depicted. Using the identifi ca- Southwest Turkey show clearly darker color tion key and referring to the distribution than those from southeastern Turkey and maps, the identifi cation of all Gnopharmia Levant. In this case, fi ne results of geometric specimens is easily possible. morphometry show that these populations Before this study, we had no knowledge on form a cluster (Fig. 2). Also DNA taxonomy the biology and on host plants of Gnophar-

Entomologie heute 27 (2015) 30 HOSSEIN RAFAEI

Fig. 2: A Position of selected landmarks for geometric morphometry on the aedeagus. B Canoni- cal variates analysis (CVA) confi rms the results of morphological and molecular studies on species delimitation within the genus Gnopharmia (after RAJAEI et al. 2013a). Abb. 2: A Lage der für die geometrische Morphometrie ausgewählten Landmarken auf dem Ae- deagus. B Die kanonische Merkmalsanalyse (CVA) bestätigt die Ergebnisse der morphologischen und molekularen Untersuchungen zur Artabgrenzung innerhalb der Gattung Gnopharmia (nach RAJAEI et al. 2013a). mia species. During collecting trips in Iran, (Amygdalus) scoparia) was confi rmed (fi rst the life history of two sympatric Gnopharmia time for the genus). This biological data con- species (G. kasrunensis and G. colchidaria) was gruently confi rm the species differentiation successfully studied. Eggs, larvae, pupae, of these two sympatric species (see Fig. 4). adults and habitats of both species were The effects of the past climate oscillations illustrated and the larval host plant (Prunus on the populations are refl ected in the pres-

Fig. 3: Ultrametric maximum likelihood (ML) tree of haplotypes with bootstrap support (upper numbers for ML analysis, lower for Neighbor-Joining analysis (NJ); 1000 replicates were used). Green shows the congruence and red shows incongruence between molecular analyses and morphological analyses (see RAJAEI et al. 2013a for details). In general, these analyses are fully in agreement with the results of the geometric morphometry. A case study of the genus Gnopharmia 31

Abb. 3: Ultrametrischer Stammbaum (Maximum likelihood, ML) der Haplotypen mit Unterstützung durch Bootstrap (Zahlen oben für die ML-Analyse, Zahlen unten für die Neighbor-Joining-Analyse (NJ); 1000 Replicas). Kongruenz (grün) und Inkongruenz (rot) zwischen molekularer und morpho- logischer Analyse (s. RAJAEI et al. 2013a). Generell decken sich die Ergebnisse dieser Analysen mit den Ergebnissen der geometrischen Morphometrie.

Entomologie heute 27 (2015) 32 HOSSEIN RAFAEI

Fig. 4: Developmental stages and habitat of Gnopharmia kasrunensis. A Eggs. B Larva. C Pupa. D Ras-Kuh, Fars province, Iran (after RAJAEI 2010). Abb. 4: Entwicklungsstadien und Habitat von Gnopharmia kasunensis. A Eier. B Larve. C Puppe. D Ras-Kuh, Provinz Fars, Iran (nach RAJAEI 2010). ent population variation and speciation. genetic data of this species. The results of These effects could be traced by comparison these two datasets fully allow to see their of the population genetics. In a part of population distributions in the past (Fig. our study the population structures of G. 6). Based on these results we were able to colchidaria and G. kasrunensis were compared. postulate a likely scenario for postglacial Different genetic indices (e.g. haplotype distribution of these species (Fig. 6). and genetic diversity) and haplotype relationships of these two Gnopharmia species 3. Conclusions indicate the existence of at least three Qua- ternary refugia in Iran and its neighboring Complex taxonomic problems are like a mul- countries. As these refugial areas match with tidimensional object, which in most cases the current distribution of the subspecies of need to multiple disciplines to be resolved. Gnopharmia colchidaria, these results conﬁ rm Looking to a multidimensional object only also our taxonomic hypothesis about this from one side will not help us to understand species (Fig. 5). its whole concept. Today a wide range of As an independent line of data, the results standard tools (traditional and geometric of species distribution modeling of Gno- morphometry, descriptive morphology, pharmia colchidaria were compared with the DNA barcoding, phylogeographical, eco- A case study of the genus Gnopharmia 33

Fig. 5: Haplotype networks of two Gnopharmia spp.: Localities are coded with colors and the diameter of the circles shows the haplotype frequency in the whole data set. High haplotype frequency of Fars and Khorasan localities conﬁ rms the existence of an old population from last ice age in these areas (see RAJAEI et al. 2013b for details). Abb. 5: Netzwerk der Haplotypen von zwei Gnopharmia spp.: Fundorte sind farbig markiert; die Durchmesser der Kreise deuten die Häuﬁ gkeit der Haplotypen im gesamten Datensatz an. Die hohe Haplotyp-Frequenz bei Fars und Khorasan bestätigt für diese Gebiete die Existenz einer alten Population aus der letzten Eiszeit (s. RAJAEI et al. 2013b für Details).

Entomologie heute 27 (2015) 34 HOSSEIN RAFAEI

Fig. 6: Species distribution models for Gnopharmia colchidaria based on the current distribution (map on the top). Projection of the current distribution to the past (6,000 and 21,000 years before present (BP) based on CCSM model system (below) conﬁ rms the existence of multiple refugia in 21,000 years BP (red circles) (see RAJAEI et al. 2013b for more details). Abb. 6: Artverbreitungsmodelle für Gnopharmia colchidaria auf Grundlage der gegenwärtigen Verbrei- tung (Karte oben). Die Projektion der gegenwärtigen Verbreitung auf die Vergangenheit (vor 6.000 und 21.000 Jahren), basierend auf einem CCSM-Modellsystem (unten), bestätigt das Vorhandensein vieler Refugien vor 21.000 Jahren (rote Kreise) (s. RAJAEI et al. 2013b für Details). logical and biogeographical methods) are Literature developed and available to increase the quantity and quality of our arguments to DARWIN, C. (1859): The origin of species support the best taxonomic hypothesis. by means of natural selection. Published In most cases, the disagreements between two in Oct. 2006 by Dover publication, Inc., methodological approaches originate from New York. overlooked, unsolved (maybe new) problems, HEBERT, P.D.N., CYWINSKA, A., BALL, S.L., & DE WAARD, J.R. (2003): Biological identiﬁ cations which need a new approach to be employed. through DNA barcodes. Proceedings of the This may open new windows for researchers. Royal Society London Series B 270: 313-321. Furthermore, using a wide range of different HENNIG, W. (1950): Grundzüge einer Theorie methods and approaches will decrease the der phylogenetischen Systematik. Deutscher probability of failure and misunderstandings. Zentralverlag, Berlin. A case study of the genus Gnopharmia 35

JAHNS, I. (ed. 2000): Geschichte der Biologie J.W. (2013b): Quaternary refugia in south- – Theorien, Methoden, Institutionen, Kurz- western Iran: Insights from two sympatric biographien. (3. Auﬂ age). Spektrum Akade- moth species and independent lines of mischer Verlag, Heidelberg, Berlin evidence. Organisms Diversity & Evolution MAYR, E. (1970): Populations, species, and 13: 409-423. evolution. An abridgment of animal species SCHLICK-STEINER, B.C., STEINER, F.M., SEIFERT, B., and evolution. Harvard University Press, STAUFFER, C., CHRISTIAN, E., & CROZIER, R.H. Cambridge, USA. (2010): Integrative taxonomy: A multisource PADIAL, J.M., MIRALLES, A., DE LA RIVA, I., & approach to exploring biodiversity. Annual VENCES, M. (2010): The integrative future Revue of Entomology 55: 421-38. of taxonomy. Frontiers in Zoology 7: 1-14. SMITH, R., RASSMANN, K., DAVIES, H., & KING, RAJAEI, SH.H. (2010): Life-history of Gnopharmia N. (eds.) (2011): Why taxonomy matters. kasrunensis Wehrli, 1939 and G. colchidaria Le- http://www.bionet-intl.org/why. BioNET- derer, 1870 (Geometridae, Ennominae) and INTERNATIONAL, Egham, UK. their distribution in Iran, with ﬁ rst host-plant SCOBLE, M.J., & HAUSMANN, A. [updated 2007]: records for the genus. Bonn Zoological Bul- Online list of valid and available names of letin 57: 65-73. the Geometridae of the World, http://lep- RAJAEI, SH.H., STÜNING, D., & TRUSCH, R. (2012): barcoding.org/geometridae/species_check- Taxonomic revision and distribution patterns lists.php of the species of Gnopharmia Staudinger, SIMPSON, G.G. (1951): The species concept. 1892 (Geometridae, Ennominae). Zootaxa Evolution 5: 285-298. 3360: 1-52. VAN VALEN, L. (1976): Ecological species, multi- RAJAEI, SH.H., STRUWE, J.F., RAUPACH, M.J., species, and oaks. Taxon 25: 233-239. AHRENS, D., & WÄGELE, J.W. (2013a): Integra- tion of cytochromec oxidase I barcodes and Dr. Hossein Rajaei geometric morphometrics to delimit species in the genus Gnopharmia (Lepidoptera: Geo- Staatliches Museum für Naturkunde metridae, Ennominae). Zoological Journal of Stuttgart the Linnean Society 169: 70-83. Rosenstein 1 RAJAEI, SH.H., RÖDDER, D., WEIGAND, A.M., D-70191 Stuttgart DAMBACH, J., RAUPACH, M., & WÄGELE, E-Mail: [email protected]

Entomologie heute 27 (2015)