DNA barcoding and molecular phylogeny of Eois moths (Geometridae) from southern Ecuador

Die Gattung Eois (Larentiinae) ist Teil einer megadiversen Fauna von Geometriden in den Bergregenwäldern des südlichen Ecuador. Angesichts der hohen Diversität an Morphotypen und der engen Beziehung zu Futterpflanzen aus der Gattung Piper wurde die Gattung Eois zur weiterführenden Untersuchung ausgewählt. Hauptfragestellungen waren die Brauchbarkeit von Barcode-Ansätzen zur Artidentifikation sowie ein erster Einblick in phylogenetische Zusammenhänge innerhalb von Eois. COI-Barcode-Sequenzen von 340 Individuen wurden generiert, um eine Datenbank der Artengemeinschaft an der Sammlungslokalität zu erstellen. Dieses Inventar wird von großem Nutzen bei der Identifikation von Raupen sein und damit die Zahl der Futterpflanzennachweise ohne die Notwendigkeit zeitraubender Zuchten erhöhen. Die Analyse der Barcoding-Sequenzen zeigte, dass es im Fall einer rein Morphologie-basierten Artabgrenzung zu einer breiten Überlappung von intra- und interspezifischen genetischen Distanzen kommt. Artabgrenzung auf Sequenzbasis mit einem Schwellenwert von 3% eliminiert 6 von 9 Fällen, die eine Überlappung verursachen. Abgrenzung mit einem 2% Schwellenwert führt zu einem klaren "barcoding gap". Es konnten allein aufgrund der Flügelzeichnungsmerkmale 30 neue Morphosspezies im vorliegenden Material identifiziert werden, deren Artstatus sich ausnahmslos durch Analyse der DNA-Barcode-Sequenzen bestätigte. Die Zahl der Eois-Arten in diesem kleinen, nur wenige Quadratkilometer großen Areal an Bergregenwald steigt daher von 99 auf 129. Diese Zahl erhöht sich weiter (auf 138), wenn 9 potentielle Arten, welche durch genetische Artabgrenzung unter Anwendung eines 2% Schwellenwertes identifiziert wurden, mit eingerechnet werden. Erweiterte COI Sequenzen (1220bp) und Elongation-factor-1-alpha Sequenzen (1066bp) von 102 Taxa wurden generiert. Maximum parsimony und Bayes'sche Methoden der Phylogenierekonstruktion wurden angewandt, um die Verwandtschaftsverhältnisse innerhalb Eois abzuschätzen. Alle Analysen bestätigten die Monophylie der untersuchten neotropischen Vertreter der Gattung Eois. Die Stammbäume zeigen, dass alle betrachteten Flügelmuster-Typen – bis auf eine Ausnahme – nur einmal entstanden sind. Diese Ausnahme betrifft den basalen Clade innerhalb Eois, welcher einem abgeleiteteren Clade ähnelt. Dies könnte den plesiomorphen Zustand repräsentieren oder ein Fall konvergenter Evolution sein. Futterpflanzenbeziehungen zu Piper sind über die gesamte Gattung hinweg zu finden. Jedoch sind die vorliegenden Nachweise auf bestimmte Subclades konzentriert. Die bislang in der Literatur vermutete nahe phylogenetische Verwandtschaft der Gattung Eois mit dem Tribus Eupitheciini (Blütenspanner) konnte anhand der molekularen Daten nicht bestätigt werden.The genus Eois (Larentiinae) comprises an important part of a megadiverse assemblage of geometrid moths in the mountain rainforests of southern Ecuador. Owing to the diversity of morphotypes found in this genus, the abundance of many of its component species, and the close relationships with larval food plants in the genus Piper, the genus Eois was chosen as a target for more detailed investigation on the suitability of DNA barcode applications and phylogenetic relationships. COI barcode sequences (676 bp) were generated from 342 individuals to establish an inventory of DNA barcodes from one study area (Reserva Biológica San Francisco). This inventory can be used to match larvae to known adult samples without rearing, and will therefore be of vital help to extend the currently very limited knowledge about food plant relationships and host specialization. Analyses of barcoding sequence divergence showed that species delimitations based on external morphology alone result in broad overlap of intra- and interspecific distances. Species delimitation at a 3% pairwise genetic distance threshold eliminated all but 3 out of 9 cases causing overlap and delimitation at a 2% threshold resulted in a clear barcoding gap. 30 previously unrecognized species were identified solely on morphological evidence, and their species status was subsequently supported by barcode sequence data. In addition to that 9 putative species could be recognized by threshold based delimitation. Hence, the present study increased the number of Eois species recorded from that small area of Andean mountain forests from 99 to 129 (morphology-based) or even 138 (sequence-based). Notably there are no cases where two or more previously known morphospecies had to be lumped. We acquired elongation factor 1-alpha sequences and extended COI sequences of 102 individuals amounting to a combined sequence dataset of ~2200 bp. These represented at least 70 Eois species plus a range of outgroup taxa. Maximum parsimony and Bayesian inference of phylogeny were employed to estimate relationships within Eois. Monophyly of Eois was always strongly supported. Phylogenetic trees revealed that all included wing-pattern types except for one evolved only once. The exception to this rule occurred in the most basal clade whose species closely resemble those in a clade higher up in the tree. This may represent an ancestral character state or the result of convergent evolution. Additionally it could be determined that species known to feed on Piper are spread over most of the major clades within Eois, indicating that the host plant relationship with Piper is indeed a widespread trait among the Neotropical members of the genus and not just occurs in certain subclades. The number of species and individuals recorded from Piper is however skewed towards two subclades. Within the subfamily Larentiinae the genus Eois has traditionally been placed close to the tribe Eupitheciini, but there is presently no support for such a placement in our phylogenetic analyses

“Into and out of” the Qinghai‐Tibet plateau and the Himalayas: centers of origin and diversification across five clades of Eurasian montane and alpine passerine birds

Encompassing some of the major hotspots of biodiversity on Earth, large mountain systems have long held the attention of evolutionary biologists. The region of the Qinghai‐Tibet Plateau (QTP) is considered a biogeographic source for multiple colonization events into adjacent areas including the northern Palearctic. The faunal exchange between the QTP and adjacent regions could thus represent a one‐way street (“out of” the QTP). However, immigration into the QTP region has so far received only little attention, despite its potential to shape faunal and floral communities of the QTP. In this study, we investigated centers of origin and dispersal routes between the QTP, its forested margins and adjacent regions for five clades of alpine and montane birds of the passerine superfamily Passeroidea. We performed an ancestral area reconstruction using BioGeoBEARS and inferred a time‐calibrated backbone phylogeny for 279 taxa of Passeroidea. The oldest endemic species of the QTP was dated to the early Miocene (ca. 20 Ma). Several additional QTP endemics evolved in the mid to late Miocene (12–7 Ma). The inferred centers of origin and diversification for some of our target clades matched the “out of Tibet hypothesis’ or the “out of Himalayas hypothesis” for others they matched the “into Tibet hypothesis.” Three radiations included multiple independent Pleistocene colonization events to regions as distant as the Western Palearctic and the Nearctic. We conclude that faunal exchange between the QTP and adjacent regions was bidirectional through time, and the QTP region has thus harbored both centers of diversification and centers of immigration

New perspectives on the evolution of Andean moths

Die tropischen Anden sind einer der ausgeprägtesten Biodiversitätshotspots der Erde. Wie diese bemerkswerte Diversität enstanden ist ist jedoch noch immer nur unzureichend bekannt. Erst in den letzen 20 Jahren wurde es durch das aufkommen von molekularphylogenetischen Methoden möglich die Evolution dieser artenreichen Ökosysteme im detail zu untersuchen. Diese Disseration bietet neue Einblicke in die Evolution von andinen Nachtfaltern. Zwei Gattungen wurden einer tiefgehenden Untersuchung hinsichtlich ihrers evolutionären Ursprungs, ihrer Systematik und ihrer Futterpflanzennutzung unterzogen. Die Gattung Eois (Geometridae) war schon lange Zeit im Fokus der Wissenschaft. Besondere Aufmerksamkeit erhalten haben diese Falter durch ihre enge Beziehung zu ihren Futterpflanzen der Gattung Piper. In der Vergangenheit wurde in der Literatur mehrfach eine reziproke Ko-evolution von Eois mit Piper Pflanzen postuliert. In dieser Arbeit wird Eois einer Neuuntersuchung mit einem doppelt so großen Artensampling unterzogen. Diese neue Analyse hat die Erkenntnisse aus bisherigen Studien im wesentlichen bestätigt. Alle zuvor innerhalb von Eois erkannten Kladen wurden bestätigt und weitere bisher unerkannte wurden gefunden. Die fossil-kalibrierte Phylogenie für Eois bestätigt das die wesentliche Radiation von Eois in der zweiten Hälfte des Miozän stattgefunden hat. Speziationsereignisse dieser Nachtfalter wurden damit vermutlich ganz wesentlich von den zeitgleich sich auffaltenden Anden beeinflusst und vorangetrieben, sowohl direkt als auch indirekt über die Diversifikation der Futterpflanzen. Eine Analyse der phylogenetischen Verteilung der Fraßpflanzennutzung hat gezeigt das mehrere evolutionäre Übergänge zu andern Pflanzen als Piper stattgefunden haben. Dabei waren sowohl Wechsel auf andere Gattung innerhalb der Piperaceae feststellbar als auch zu anderen Pflanzenfamilien. Diese Ergebnisse dienen als sehr starke Indikation dafür das Eois nicht in einem Ko-Evolutionsszenario mit Piper involviert ist. Ko-Evolution mit Piper ist, sofern überhaupt vorhanden, daher auf kleinere Artengruppen innerhalb von Eois beschränkt. Weiters werden erste Erkenntnisse zu einer weiteren artenreichen andinen Gattung von Geometriden vorgestellt. Die Gattung Psaliodes wurde bisher nicht in einem phylogenetischen Kontext untersucht. Psaliodes Falter sind vor allem bekannt durch ihre Assoziation mit Farnen als Futterpflanze. Die Nutzung von Farnen ist innerhalb der Geometridae sehr selten. Die vorhandenen Fraßnachweise für Psaliodes weisen darauf hin das die Tiere bei der Wahl der Fraßplanze vergleichsweise uneingeschränkt zwischen den verschiedensten Farnen wählen können. Einzelne Arten von Psaliodes befressen bis zu fünf Arten von Farnen aus unterschiedlichsten Familien. Die Ergebnisse der Analyse von Psaliodes hat gezeigt das Psaliodes nicht monophyletisch ist, es wurde sehr starke Ünterstützung für eine Aufspaltung von Psaliodes in zwei nicht näher verwandte Kladen gefunden. Bemerkenswert ist das beiden dieser Kladen farnfressende Taxa angehören. Dies impliziert daher das in den Neotropischen Geometridae die Nutzung von Farnen zweimal unabhängig enstanden ist. Wie auch bei Eois erkannt wurde fanden auch im Fall von Psaliodes die wesentlichen Artbildungsereignisse in der zweiten Hälfte des Miozän statt. Die lokale Nachtfalterfauna der Reserva Biologica San Francisco im südlichen Ecuador hat bereits als Modell für Untersuchungen hinsichtlich Umwelt- und Höhengradienten gedient. In dieser Dissertation wird diesen Daten eine evolutionäre Perspektive beigestellt. Über 3000 Arten von Geometriden wurden in einem community-phylogeny Ansatz analysiert. Es hat sich herausgestellt das Artengemeinschaften die in großen Höhen vorkommen zwar ein moderates jedoch kein besonders ausgeprägtes Ausmaß an phylogenetischer Klumpung aufweisen während Gemeinschaften in geringen Höhen eine phylogenetische Überdispersion zeigen. Es kann daher davon ausgegangen werden das ein sogenanntes „environmental filtering“ eine eher untergeordnete Rolle gespielt hat und diese Nachfalter ungeachtet ihrer phylogenetischen Affinität sehr gut adaptiert sind um neue Lebensräume zu besiedeln. Im letzten Kapitel wird über die Entwicklung und Verifizierung von Methoden zur Gewinnung von DNA barcodes aus bis zu 157 Jahren alten Typenexemplaren von Eois berichtet. Es konnten dabei selbst von den ältesten Tieren erfolgreich Sequenzen gewonnen werden. Diese Daten sind von unschätzbarem Wert für zukünftige taxonomische Revisionen und der Beschreibung von neu entdeckten Arten.The tropical Andes are one of the foremost biodiversity hot-spots on the planet. How this exceptional biodiversity came into existence is still only incompletely known. It was only in the last 20 years with the advent of molecular phylogenetics that a detailed exploration of those evolutionary mechanisms became possible. This dissertation provides new insights into the evolution of Andean moths using molecular phylogenetic tools and provides tools to facilitate future research on those megadiverse communities. Two genera of moths (Geometridae) were subjected to in-depth studies of past evolutionary dynamics, evolution of larval host-plant use, and their systematics. The genus Eois (Geometridae) has already been in the focus of investigations of environmental gradients, species diversity and especially its host-plant associations with Piper plants. Close co-evolution of Eois and Piper has repeatedly been proposed in the literature. This dissertation provides an updated molecular phylogeny for Eois with a more than two-fold increase in taxon sampling. This new analysis confirmed findings of a previous molecular phylogenetic study on Eois. All previously recognized infrageneric clades within Eois could be confirmed and new clades were detected. The time calibrated phylogenetic hypothesis confirmed that Neotropical Eois diversified in the second half of the Miocene. Speciation in Eois was therefore likely mediated through the Andean uplift, both directly as well as indirectly through contemporary diversification of their hostplants. Analysis of phylogenetic patterns of hostplant use revealed several evolutionary switches to plants other than Piper, both the other genera within Piperaceae as well as to entirely different plant families. Those result suggest that Eois is not involved in a large-scale co-evolutionary scenario with Piper plants. Co-evolution with Piper is, if at all present, limited to certain clades and species groups within Eois. First insights are provided into the evolution of a second species rich Andean genus of geometrid moths, the genus Psaliodes. This genus was not previously subjected to a molecular or even morphological phylogenetic analysis. Psaliodes moths are known to feed on ferns which is a very rare trait among Geometridae and even more so among the subfamily Larentiinae which Psaliodes belongs to. As far as known there is only one other major radiation I fern feeding Geometridae besides Psaliodes. Host plant records for Psaliodes indicate that those moths are comparatively unconstrained in their selection of larval hosts among ferns, single species of Psaliodes feed on up to five families of ferns. The results on Psaliodes revealed that the genus is most likely not monophyletic, strong evidence was recovered that this genus is actually a polyphyletic assemblage of two not particularly closely related clades. Even more striking is that both of those clades contain fern feeding taxa, implying that fern feeding evolved twice independently in Neotropical Geometridae. As is the case for Eois, Psaliodes diversified in the second half of the Miocene, thereby adding to an increasing amount of evidence emphasizing the role of the Andes for the origin of Neotropical biodiversity. The local moth fauna in a section of montane rainforest at the Reserva Biologica San Francisco in southern Ecuador has served as a model system for studies on elevational and environmental gradients. Here a novel perspective on those previously obtained results is provided. Over 3000 species of geometrid moths were analyzed in a community phylogeny approach in order to detect elevational patterns of phylogenetic diversity. It was found that high elevation communities exhibit a moderate amount of phylogenetic clustering compared to low elevations communities. Environmental filtering has therefore likely played a subordinate role and geometrid moths are apparently not strongly constrained when colonizing new habitats. In the final chapter of this dissertation crucial tools for taxonomy and biodiversity research were developed and tested. The inadequate taxonomic treatment of Neotropical taxa is a major impediment for research. Sharing of results among different researchers is tedious or even entirely impossible as the taxonomic identity of the studied taxa can not be verified. It is therefore imperative to work towards taxonomic revisions and description of newly discovered species to abolish the need for countless provisional classifications. This thesis provides a case study and a newly developed toolkit for the acquisition of DNA barcodes from primary type specimens

K (2011) DNA barcoding-based species delimitation increases species count of Eois (Geometridae) moths in a wellstudied tropical mountain forest by up to 50%. Insect Sci 18

Abstract The genus Eois comprises an important part of megadiverse assemblages of geometrid moths in mountain rainforests of southern Ecuador. In this study we report: (i) on the construction of a DNA barcode library of Eois for identification purposes; and (ii) the exploration of species diversity through species delimitation by pair-wise distance thresholds. COI barcode sequences were generated from 408 individuals (at least 105 species) collected on a narrow geographic scale (∼40 km 2 ) in the Reserva Biológica San Francisco. Analyses of barcode sequence divergence showed that species delimitations based solely on external morphology result in broad overlap of intra-and interspecific distances. Species delimitation at a 2% pair-wise distance threshold reveals a clear barcoding gap. Fifty-two previously unrecognized species were identified, 31 of which could only be distinguished by an integrative taxonomy approach. Twelve additional putative species could only be recognized by threshold-based delimitation. Most splits resulted in two or three newly perceived cryptic taxa. The present study increased the number of Eois species recorded from that small area of Andean mountain forest from 102 to 154 (morphology-plus integrative taxonomy-based) or even 166 (sequence-based), leaving the species accumulation curve still far from reaching an asymptote. Notably, in no case did two or more previously distinguished morphospecies have to be lumped. This barcode inventory can be used to match larvae to known adult samples without rearing, and will therefore be of vital help to extend the currently limited knowledge about food plant relationships and host specialization

DNA barcode sequencing from old type specimens as a tool in taxonomy: a case study in the diverse genus Eois (Lepidoptera: Geometridae).

In this study we report on the sequencing of the COI barcode region from 96 historical specimens (92 type specimens +4 non-types) of Eois. Eois is a diverse clade of tropical geometrid moths and is the target of a number of ongoing studies on life-histories, phylogeny, co-evolution with host plants or parasitoids, and diversity patterns across temporal and spatial dimensions. The unequivocal application of valid names is crucial for all aspects of biodiversity research as well as monitoring and conservation efforts. The availability of barcodes from historical type specimens has the potential to facilitate the much-needed acceleration of species description. We performed non-destructive DNA extraction on the abdomens of Eois specimens between 79 and 157 years of age. We used six primer combinations (recovering between 109 and 130 bp each) to target the full-length barcode sequence of each specimen. We were able to obtain sequences for 91 of 96 specimens (success rate 94.8%). Sequence length ranged from 121 bp to full barcode sequences (658 bp), the average sequence length was ~500 bp. We detected a moderately strong and statistically significant negative correlation between specimen age and total sequence length, which is in agreement with expectations. The abdomen proved to be an exceedingly valuable source of DNA in old specimens of Lepidoptera. Barcode sequences obtained in this study are currently being used in an effort towards a step-wise taxonomic revision of Eois. We encourage that DNA barcodes obtained from types specimens should be included in all species descriptions and revisions whenever feasible

The trinity of ecological contrasts: a case study on rich insect assemblages by means of species, functional and phylogenetic diversity measures

Abstract Background The ‘classical’ concept of species diversity was extended in the last decades into other dimensions focusing on the functional and phylogenetic diversity of communities. These measures are often argued to allow a deeper understanding of the mechanisms shaping community assembly along environmental gradients. Because of practical impediments, thus far only very few studies evaluated the performance of these diversity measures on large empirical data sets. Here, data on species-rich riparian moth communities under different flood regimes and from three different rivers has been used to compare the power of various diversity measures to uncover ecological contrasts. Results Contrary to the expectation, classical metrics of species diversity (Hill numbers N1, N2 and Ninf) and evenness (Buzas-Gibson’s E and Pielous’s J) turned out to be the most powerful measures in unravelling the two gradients investigated in this study (e.g. flood regime and region). Several measures of functional and phylogenetic diversity tended to depict either only one or none of these contrasts. Rao’s Q behaved similarly as species diversity and evenness. NTI and NRI showed a similar pattern among each other but, were different to all the other measures. Functional Divergence also behaved idiosyncratically across the 28 moth communities. The community weighted means of nearly all individual functional traits showed significant ecological patterns, supporting the relevance of the selected traits in shaping assemblage compositions. Conclusions Species diversity and evenness measures turned out to be the most powerful metrics and clearly reflected both investigated environmental contrasts. This poses the question when it is useful to compile the additional data necessary for the calculation of additional diversity measures, since assembling trait bases and community phylogenies often requires a high work load. Apart from these methodological issues, most of the diversity measures related to communities of terrestrial insects like moths increased in forests that still are subject to flooding dynamics. This emphasizes the high conservation value of riparian forests and the importance of keeping and restoring river dynamics as a means of fostering also terrestrial biodiversity in floodplain areas

Sequence length distribution.

<p>Chart showing the frequency distribution of total sequence lengths obtained from type specimens of Neotropical <i>Eois</i> moths.</p

Sequence length vs. specimen age: Scatterplot of specimen age versus total obtained sequence length.

<p>A linear regression line has been fitted to the data; R² = 0.148, p<0.001.</p

PCR and sequencing primers: List of PCR and sequencing primers with the expected length of each amplicon indicated.

<p>PCR and sequencing primers: List of PCR and sequencing primers with the expected length of each amplicon indicated.</p