Sexual Dimorphism and Reproductive Phenology of Common Birds in São Tomé Island – Conservation Implications

UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE BIOLOGIA ANIMAL

Sexual dimorphism and reproductive phenology of common birds in São Tomé Island – conservation implications

Bárbara de Castro Marques Arez Madeira

Mestrado em Biologia da Conservação

Dissertação orientada por:

Doutor Ricardo Faustino Lima

Doutor Martim Pinheiro de Melo

2018

AGRADECIMENTOS

Quero começar por agradecer aos meus orientadores por todo o apoio que me deram quer em São Tomé, quer no Porto e em Lisboa, nunca deixei de me sentir apoiada. Quero agradecer principalmente ao Ricardo Lima pela ajuda e orientação em São Tomé. Sem ele seria muito difícil orientar o trabalho de campo e toda a logística que isso envolve, pelo incansável apoio ao longo do ano e por me incentivar mesmo quando parecia difícil avançar. Quero também agradecer a duas pessoas impecáveis ao Martim Melo e à Rita Covas por me receberem em sua casa como se eu fosse família e me orientarem no laboratório para o tratamento das amostras de ADN, pela sua disponibilidade em fazer reuniões mesmo estando longe ou atarefados, agradeço imenso os vossos “inputs” para melhorar o desenvolvimento da tese e por vezes “desatar nós” quando parecia não haver solução.

Quero também agradecer a simpatia e a ajuda determinante de Carlos Pacheco, e a sua disponibilidade para viajar para São Tomé, onde ajudou na identificação de algumas características morfológicas das nossas espécies em estudo. Com a sua ajuda pudemos também confirmar algumas suspeitas e ideias.

O trabalho de campo foi parcialmente financiado por uma “Booster Grant” da “Rufford Foundation” (“The Role of Bird Seed Dispersal on São Tomé Forest Dynamics” - Ref.: 18618-B).

Quero agradecer em especial ao Eng. Arlindo Carvalho, Director Geral do Ambiente por apoiar as nossas actividades em São Tomé e nos dar as autorizações necessárias para o desenrolar das mesmas.

Agradeço ainda à Associação Monte Pico, pelo alojamento durante a minha estadia em São Tomé e pela ajuda no trabalho de campo.

Agradeço também aos meus incansáveis guias de campo, Octávio Veiga, Leonel Viegas e Sedney Samba que conseguiam levar quilos de carga para os acampamentos que ficavam a uns bons metros senão quilómetros das estradas, por terras para mim escorregadias que para eles eram do mais estável que se arranjava. Um especial obrigado ao Octávio pela sua hospitalidade, companhia nos finais de tarde no campo, pelas suas piadas e histórias e claro por ser um verdadeiro chefe, cozinhando as suas fantásticas refeições numa fogueira, não era de todo algo que estivesse habituada.

Tenho de agradecer a todas as pessoas que conheci em São Tomé, pois ajudaram-me a abrir os horizontes. Permitiram-me “espreitar” para as suas vidas e a experienciar durante dois meses a sua cultura, comida e costumes. Agradeço do fundo do coração à família que mais nos recebeu em sua casa e um imenso obrigada à Lucy, que nos recebeu em casa como se fossemos filhas, pelas refeições cinco estrelas, e sobretudo pelo apoio emocional, sem ti São Tomé não teria sido a mesma coisa, nunca esquecerei o teu sorriso terno.

Agradeço ao Gégé Lima por ser imparável e a pessoa mais prestável que conheci sempre pronto a ajudar na anilhagem, por nos levar a conhecer a as magnificas paisagens e cascatas escondidas da ilha e com uma simpatia do mais genuíno que poderemos encontrar deixei um verdadeiro amigo naquela ilha.

Obrigada ao Catoninho ao Lito e Lau pela sua boa disposição e carinho demonstrado ao longo da nossa visita.

Um obrigado especial à minha mãe que sempre me apoiou em todas as minhas decisões. Quero agradecer ao Rui, que para além de me ajudar a perceber algumas coisas quando era preciso, teve de me aturar a falar de coisas que não entendia muito bem, e de dar a volta, no sentido de me ajudar, quer na resolução da tese, quer emocionalmente. De tal modo que já não quer ouvir falar de aves nos próximos meses. Obrigado por estares cá sempre para mim.

Finalmente gostaria de agradecer a todos os meus amigos e colegas de mestrado pelo apoio principalmente ao Fernando e Manuel, e um obrigado especial à Filipa e Martina pela companhia nesta aventura partilhada em São Tomé, e pelo ombro amigo em Portugal, vão estar sempre na minha memória.

III

RESUMO ALARGADO

As ilhas são reconhecidas como importantes para a biodiversidade e como tal são frequentemente reconhecidas como áreas prioritárias para a conservação. Devido ao isolamento e à maior simplicidade das relações ecológicas, as ilhas também são frequentemente utilizadas como modelos para estudos ecológicos. Nesta tese tentamos complementar alguns estudos realizados ao longo dos anos sobre a importante comunidade de aves da ilha de São Tomé, caracterizada por um elevado grau de endemismo. Os dois capítulos investigam as diferenças de coloração e massa entre os machos e as fêmeas e a fenologia das espécies mais comuns de São Tomé, uma informação crucial tanto para pesquisas fundamentais sobre ecologia como para a conservação desta comunidade única.

São Tomé, com uma área de 857 km2, é a maior ilha da República Democrática de São Tomé e Príncipe, no Golfo da Guiné. Encontra-se a 255 km do continente africano e a 150 km da ilha do Príncipe. A 2 024 m de altitude o Pico é o ponto mais alto e explica o acentuado gradiente climático da ilha, caracterizado por altos níveis de humidade e chuvas frequentes no sudoeste da ilha, que contrastam com o ambiente semiárido do nordeste. Originalmente coberta por floresta, hoje em dia a floresta nativa está sobretudo circunscrita às áreas montanhosas do centro e sudoeste da ilha. Esta está envolta por floresta secundária, plantações de sombra e zonas não florestadas, que na sua maioria resultaram de uso agrícola e expansão urbana. São Tomé é extremamente importante para a conservação de aves a nível global, possuindo 17 espécies endémicas, além de três espécies partilhadas apenas com as outras ilhas do Golfo da Guiné. Com o rápido desenvolvimento económico da ilha nos últimos anos, tornaram-se ainda mais necessários esforços activos para a conservação das suas espécies e ecossistemas.

Determinar o sexo e a idade dos indivíduos em populações selvagens é importante para estudos ecológicos e para conservação. No entanto, para muitas espécies de aves, sexo e idade são difíceis de determinar, por isso decidimos abordar este tema no primeiro capítulo desta tese de forma a poder melhorar o conhecimento sobre as diferenças entre o sexo e a idade das espécies mais comuns existentes na ilha de São Tomé.

Os ambientes insulares oferecem oportunidades únicas para estudar a evolução do dimorfismo sexual porque as populações insulares frequentemente enfrentam diferentes pressões de predação, condições de alimentação, competição intraespecífica e interespecífica do que as populações continentais. O objectivo principal deste capítulo foi o de obter um melhor conhecimento do dimorfismo sexual das espécies de aves de São Tomé. Medições retiradas no campo, fotografias e sexagem molecular de 1046 indivíduos, permitiram identificar características relacionadas ao sexo e à idade para as 12 espécies mais frequentemente capturadas em redes de anilhagem. Oito dessas espécies eram sexualmente dimórficas, enquanto as quatro restantes não podiam ser sexadas com base em caracteres observáveis. O uso de modelos lineares generalizados baseados em biometria para desenvolver funções discriminantes baseadas em morfometria permitiu distinguir o sexo de três destas espécies. A incorporação de informações sobre altitude e tipo de habitat permitiu ainda distinguir o macho e a fêmea dos Tecelões de São Tomé Ploceus sanctithomae, enquanto melhorou a capacidade de distinguir o sexo da maioria das outras espécies. A coloração foi crucial para identificar a idade na maioria das espécies e contribuiu para a sexagem de adultos em seis espécies, em duas das quais apenas os machos adultos puderam ser identificados com um alto grau de confiança.

Os organismos evoluíram para coordenar os seus esforços reprodutivos com o pico de recursos necessários para criar as suas crias. Em zonas temperadas, isso levou a épocas de reprodução curtas

IV coincidentes com uma sazonalidade marcada na disponibilidade de recursos. Em contraste, as áreas tropicais próximas ao Equador são alguns dos ambientes mais estáveis da Terra, com pouca sazonalidade na disponibilidade de recursos. Isso sugere que as espécies da floresta tropical se possam reproduzir durante a maior parte do ano. Por outro lado, a variação na precipitação pode levar a mudanças na disponibilidade de recursos que afectam as decisões reprodutoras. A insuficiência de estudos detalhados sobre a época de reprodução das espécies da floresta tropical, não permitiu testar isso efectivamente, mesmo em aves - um dos grupos de vertebrados mais bem estudados. Neste segundo capítulo realizou-se um estudo detalhado sobre a sazonalidade de reprodução e muda das espécies de aves comuns de São Tomé, e a sua associação com variáveis ambientais e características de espécies. As épocas de reprodução e muda foram identificadas para cada espécie com base em dados de anilhagem para as 12 espécies com o maior número de registos. A época de reprodução das espécies de aves comuns de São Tomé ocorreu principalmente durante a estação das chuvas, atingindo o pico durante a estação seca curta (gravanito). A muda seguiu-se á época de reprodução, e ainda assim ocorreu antes do início da longa estação seca, a gravana. As espécies mais pesadas, com períodos de reprodução mais longos, tendem a reproduzir-se mais cedo de forma a garantir que as eclosões coincidam com o pico de alimento. Os nossos resultados também sugerem que a muda, e portanto a reprodução, são mais tardias em altitudes mais elevadas.

Este estudo melhorou muito a nossa capacidade de identificar o sexo das aves endémicas de São Tomé no campo, uma informação crucial tanto para pesquisas fundamentais sobre ecologia e evolução como para a conservação desta comunidade única. Contribuir para uma melhor compreensão da sazonalidade em São Tomé é fundamental para apoiar estratégias de conservação de aves, nomeadamente no que se refere à caça e outras actividades extractivistas. Assim forneceremos informações fundamentais para orientar acções de conservação baseadas em evidências tão necessárias para a ilha e para ecossistemas de ilhas tropicais semelhantes, uma vez que não se sabe muito sobre como as aves tropicais lidam com a variação da sazonalidade reprodutora. Também mostra que, apesar da reduzida variabilidade ambiental, o ciclo de vida anual das aves tropicais pode ser determinado pelas variações na precipitação que deverão controlar a disponibilidade de recursos.

Palavras-chave: África, endemismo, estratégias reprodutoras, florestas tropicais, funções discriminantes

ABSTRACT

The island of São Tomé is the largest of the Democratic Republic of São Tomé and Principe, in the Gulf of Guinea, central Africa. Home to a wide variety of endemic and endangered species, it is often recognized as an important area for the conservation of biodiversity worldwide, and for birds in particular. The main goal of this study was to gain a better knowledge of the sexual dimorphism and breeding seasonality of São Tomé bird species. To do so, we used and extended a bird ringing dataset, which was created in 2002. Biometrics, photographs and molecular sexing, allowed identifying characteristics related to sex and age in 12 species. Eight of these were sexually-dimorphic, while the remaining could not be confidently sexed based on observable characters. Coloration was crucial to identify age in most species, and contributed to sex adults in six species of which in two only adult males could be identified with a high degree of confidence. The development of morphometric-based discriminant functions using generalized-linear models based on biometrics allowed a better sex differentiation of three of the eight species. The incorporation of information on altitude and habitat

V type further allowed sexing the São Tomé Weavers Ploceus sanctithomae, while improving the ability to distinguish the sex of most other species. The data on brood patches and moulting of primary flight feathers allowed understanding the seasonality of breeding in the common birds of São Tomé. The breeding activity occurred mostly during the rainy season, peaking during the short dry season of gravanito. Moult followed breeding activity, and still occurred mostly before the start of the long dry season, the gravana. Heavier species tended to breed earlier, probably due to having longer breeding seasons and therefore needing to ensure that hatching coincides with peak food availability. Results show that, despite reduced environmental variability, the annual life cycle of tropical birds might be structured around precipitation-driven changes in resource availability. Our results also suggest that moult, and thus breeding, is delayed at higher altitudes. This study helped improve the knowledge of the sexual dimorphism of São Tomé bird species which is crucial for fundamental research on ecology and evolution and for the conservation of this unique community. Our phenology data are key to support bird conservation strategies, namely regarding hunting and other extractive activities and contributes towards a better understanding of seasonality on São Tomé and for similar tropical island ecosystems - since not much is known on how tropical birds cope with seasonal variations in their environment.

Keywords: África, discriminant function, endemism, reproductive strategies, tropical forests

TABLE OF CONTENTS

GENERAL INTRODUCTION……………………………………………………………………….…1

CHAPTER 1: Coloration and morphology of endemic birds for sex and age identification…………...5

INTRODUCTION……………………………………………………………………………….…..5

METHODS…………………………………………………………………………………….….…7

Study Area………………………………………………………………………………………..7

Data Collection………………………………………………………………….……..…………8

Data analysis…………………………………………………………………….………………..9

RESULTS……………………………………………………………………………………………9

Morphometrics and sex…………………………………………………………………………10

Distinguishing sex and age: Individual species accounts……………………………………….12

Morphometric-based sex discriminant functions……………………………………………….18

DISCUSSION………………………………………………………………………………………21

Age identification……………………………………………………………………………….21

Coloration………………...…………………………………………………………………..…21

Moult……………………………………………………………………………………………22

Sexing ……………………….………………………………………………………………….22

Sexual dimorphic species ………………………………………………………………………23

Non sexual dimorphic species…………………………………………………………………..24

Sex-discriminant functions analyses……………………………………………………………24

Morphometrics …………………………………………………………………………………25

Conclusions……………………………………………………………………………..……....25

CHAPTER 2: Seasonality in the tropics: Breeding and moulting of the common birds of São Tomé Island……………………………………………………………………………………………….…..27

INTRODUCTION…………………………………………………………………………….……27

METHODS…………………………………………………………………………………………29

Study Area……………………………………………………………………………………....29

Data Collection……………………………………………………………………………….....30

VII

Data analysis…………………………………………………………………………………….30

RESULTS…………………………………………………………………………………………..31

Breeding and moulting activity…………………………………………………………………33

Species traits and seasonality…………………………………………………………………...35

Environmental characteristics and seasonality………………………………………………….35

DISCUSSION………………………………………………………………………………………37

Breeding seasonality…………………………………………………………………………….37

Moulting seasonality……………………………………………………………………………38

Phenological determinants: environmental characteristics and species traits…………………..39

Implications for conservation…………………………………………………………………...39

FINAL CONSIDERATIONS………………………………………………………………………….41

REFERENCES………………………………………………………………………………………...42

SUPPLEMENTARY MATERIALS ………………………………………………………………….49

VIII

LIST OF TABLES

Table 1.1 - List of São Tomé common birds species………………………………………………….10

Table 1.2 - Measurements of São Tomé common bird species……………………………………….17

Table 1.3 - Sex and age distinctiveness of São Tomé common bird species, based on morphometrics and coloration………………………………………………………………………………………….18

Table 1.4 - Sex discriminant functions using morphometric-based GLMs for the São Tomé common bird species…………………………………………………………………………………………….19

Table 1.5 - Sex discriminant functions using environmental variables-based GLMs for the São Tomé common bird species…………………………………………………………………………………..20

Table 2.1- List of the common birds of São Tomé Bird characteristics………………………………31

Table 2.2 – Chi-square test results from stepwise backward elimination procedure on the full model and respective AIC……………………………………………………………………………………..36

LIST OF FIGURES

Figure 1.1- Map of São Tomé Island in relation to mainland Africa………………………….……….8

Figure 1.2-Morphometrics in São Tomé common bird species……………………………………….11

Figure 1.3- Photos of Newton Sunbird………………………………………………………………..12

Figure 1.4- Photos of Lemon Dove……………………………………………………………………12

Figure 1.5 - Photos of Principe Seedeater……………………………………………………………..13

Figure 1.6 - Photos of Giant Sunbird………………………………………………………………….13

Figure 1.7 - Photos of Common Waxbill……………………………………………………………...13

Figure 1.8 - Photos of Giant Weaver………………………………………………………………….14

Figure 1.9 – Photos of São Tomé Weaver…………………………………………………………….14

Figure 1.10 - Photos of São Tomé Prinia…………………………………………..………………….15

Figure 1.11 - Photos of São Tomé Paradise-flycatcher……………………………………………….15

Figure 1.12 - Photos of São Tomé Thrush…………………………………………………………….16

Figure 1.13 - Photos of São Tomé white-eye……………………...………………………………….16

Figure 1.14 - Photos of Black-capped Speirops……………………………………………………….17

Figure 2.1 - Map of São Tomé Island with sample location…………………………………………..32

Figure 2.2 - Link between rainfall, breeding and moulting seasonality in São Tomé………………...33

Figure 2.3 - Breeding and moulting activity of the 12 common bird species of São Tomé…………..34

Figure 2.4 - Correlation between the timing of breeding/moulting, and species traits………………..35

Figure 2.5- The monthly proportion of moults in altitude for the eight species with most records…..35

Figure 2.6 - All possible linear models used to explain the proportion of moulting individuals……..36

Figure S1. - Bi-plots of five morphological measurements for 11 endemic bird species of São Tomé and for one non-endemic…………………………………………………………………………...49-50

Figure S2. - São Tomé Thrush differences in iris colour between males and females of molecularly tested individuals……………………………………………………………………………………....51

Figure S3. - Brood patch sample effort distribution, throughout the year…………………………….52

Figure S4. - Moult sample effort distribution, throughout the year…………………………………..53

Figure S5. - Breeding and moulting activity of sexed females of the 12 common bird species of São Tomé…………………………………………………………………………………………………...54

LIST OF ABBREVIATIONS AND ACRONYMS

LC Least Concern VU Vulnerable NT Near threatened Ananew Anabathmis newtonii Newton Sunbird Collar Columba (larvata) simplex Lemon Dove Criruf Crithagra rufobrunnea Príncipe Seedeater Dretho Dreptes thomensis Giant Sunbird Estast Estrilda astrild Common Waxbill Plogra Ploceus grandis Giant Weaver Plosan Ploceus sanctithomae São Tomé Weaver Primol Prinia molleri São Tomé Prinia São Tomé Paradise- Teratr Terpsiphone atrochalybeia flycatcher Turoli Turdus olivaceofuscus São Tomé Thrush Zosfea Zosterops feae São Tomé White-eye Zoslug Zosterops lugubris Black-capped Speirops

GENERAL INTRODUCTION

Reproduction is a fundamental feature of life. When food resources are scarce, the climate becomes hostile, or individual survival is jeopardized by some other adverse change in living conditions, species have to employ strategies to ensure the survival of their progeny (Hau et al. 2008). To study these strategies we first need to know how to differentiate the sexes of the study species.

Determining the sex of individuals in natural populations is a useful tool for studying population dynamics, population structure, habitat use, behavior and mating systems, species vulnerability to extinction, distribution shifts and therefore for making appropriate management decisions, both at the habitat and species level (Shaw et al. 2003; Schwartz 2012). Sexual dimorphism is a common, and often substantial form of intraspecific phenotypic differentiation, such as in colour, shape, size, structure or behavior. It can occur in any group of living beings, from protists to plants and animals (Punzalan & Hosken 2010). When interspecific competition is small, sexes tend to diverge morphologically. In some plants, the dissimilarities are only functional, not competitive (McGee & Wainwright 2013). In birds, as in other vertebrates, the sexes usually differ in size and colour, and sometimes also in the proportion of body parts (Selander 1966). Variation in the extent of sexual dimorphism among bird species is traditionally attributed to divergences in social mating system (Owens & Harley 1998). Many birds show some dimorphism in terms of coloration, usually the female being cryptically coloured to remain hidden on the nest, while the more-colourful male uses it to for territorial and courtship behaviors (McGee & Wainwright 2013). Unfortunately, for many bird species it is difficult to determine sex based solely on morphometrics and coloration (Calabuig et al. 2011). Molecular sexing is one of the simplest and most used DNA-based techniques applied to wild birds. These are usually applied to blood samples, although feather samples have also been used (Harvey et al. 2006). Morphometric-based sexing depends on measuring variances in body size between sexes, but its reliability can be limited and it decreases when sex dimorphism is reduced (Volodin et al. 2015). With a better knowledge of differences between sexes we can study the seasonality of breeding.

Almost all organisms experience seasonal variations in their environment conditions, such as rainfall, temperature, food abundance, disease and social factors (Nelson & Demas 2002). For birds, rainfall regimes and associated environmental changes are common cues to determine breeding seasonality (Karr 1976). Bird populations may present very different breeding seasonality depending on the region, habitat, and more specifically on the climate variations of the region they occupy (Hau et al. 2008). Breeding can be detected by changes in behavior, plumage and physiology, but the presence of a brood patch is one of the easiest and most reliable means to confirm breeding activity (Tucker 1943). The brood patch is a featherless, reddish and wrinkled skin with many blood vessels visible on the underparts of breeding female in most bird species, usually developing in the ventral region before incubation to improve heat transfer to the eggs (Demongi 2016).

Breeding and moulting, are the primary periods of the annual cycle in most birds and they are usually connected. Both actions are costly in terms of energy, and therefore they usually do not overlap in time (Demongin 2016; Freed & Cann 2011). Feather moulting is a regular replacement of all or parts of the plumage. It occurs in all species to ensure that plumage function is maintained, and it is also used to adjust plumage characteristics to specific life cycle needs of the bird (Jenni & Winkler, 1994). It should not be confused with plumage replacement, which occurs sporadically when some feathers are lost or damaged.

Compared to mainland counterparts, island birds tend to have lower fecundity, greater reproductive investment and extended developmental periods (Covas 2011). Little is known about the extent and variability of the breeding season. Despite these parameters being vital to understand population dynamics and to assess the causes behind demographic changes (Burr et al. 2016), the effort to comprehend the variation of seasonality within tropical populations and among species in tropical assemblages has been quite small (Stouffer et al. 2013).

There is now compelling evidence that the ongoing climate change is having ecological impacts on both animals and plants, from polar to tropical environments (Bellard 2012). Climate change itself is expressed in a different way across and even within tropical islands, but several factors can be recognized in tropical island ecosystems such as: climate is changing rapidly for tropical islands and that it can be expressed as a wide diversity of threats, including altered rainfall, the frequency and intensity of big storms, warming, and interactions with disturbances such as fire and invasive species (Keener et al. 2012). The responses of both flora and fauna span an array of ecosystems and organizational hierarchies, from molecular to global levels (Walter et al. 2002). The implications of climate change for birds have only recently begun to be addressed (Crick 2004). Climate affects not only the metabolic rate of birds, but also behavior, either directly or indirectly. Reproduction is a vulnerable phase of life cycle, and is thus being particularly affected by climate change. For example, it can influence the ability to carry out essential behaviors, such as courtship, while also impacting on breeding success through food scarcity, earlier breeding, changes in timing of migration and differential selection between components of a population (Crick 2004). The effects of climate change are particularly severe in tropical islands, where a large part of the species is endemic (Keener et al. 2012).

Compared to the mainland, island ecosystems are in many ways simplified (Whittaker & Palacios 2007). Islands have a smaller number of species compared with the mainland regions and have been studied for their capacity to offer insights into evolutionary and ecological processes (Roulin & Salamin 2010). This study will focus on São Tomé Island, with the goal of improving our knowledge on its birds so that insular patterns can be inferred and compared with the mainland communities.

São Tomé is an 857 km2 oceanic island about 255 km from mainland Africa. Together with Príncipe, it constitutes the Democratic Republic of São Tomé and Príncipe, in the Gulf of Guinea, Central Africa. It is a volcanic island, never connected to the mainland. Volcanic activity persisted until recently which explains the rugged topography. The highest elevation point at 2024 m a.s.l. is the “Pico de São Tomé” (Salgueiro & Carvalho 2001). The geographic location of São Tomé has allowed the development of high levels of endemism, being far enough to be isolated, while at the same time allowing a fairly high number of potential colonizers (Peet & Atkinson 1994). The mountainous center of the island halts the dominant southwest winds, creating a very strong rainfall gradient across the island: annual precipitation varying from less than 600 mm in the northeast to over 7,000 in the southwest (Bredero et al. 1977). The montane rivers have plenty of waterfalls, carving deep valleys in the center of the island, and slowing down near the coast to create small estuaries, occasionally bordered by mangroves.

Despite its proximity to the Equator, São Tomé has two clear main seasons: the dry season, between June and August, called “gravana”, and a rainy season, from September to May. There is also the “gravanito”, a smaller and less marked dry season between December and January (Tenreiro 1961). The average temperature is mostly constant throughout the year, ranging between 18°C and 33°C across the island (Bredero et al. 1977), decreasing with rainfall, altitude and in the dry season.

The humidity levels and cloud cover are also very high throughout the year and mostly towards the southwest (Carvalho et al. 2004).

The human occupation of São Tomé started in the late 15th century, when the Portuguese discovered the island, allegedly uninhabited and almost entirely covered in forest, except for tiny areas of sand dune (Peet & Atkinson 1994). The forests have a dense canopy, with trees often exceeding 30 m of height, plentiful lianas and epiphytes, like mosses, ferns and orchids (Peet & Atkinson 1994). There are three key distinct types of forest: Lowland, which extends from the sea level up to 800 m,; Montane, which extends from 800 m to 1,400 m, is composed by a dense canopy and by tall trees covered by epiphytes, and it is currently threatened by agricultural development; and Mist, which extends from 1,400 m to 2.024 m (Exell 1944), has an open canopy, the lowest temperatures and a difficult access, which has kept it protected from most human activities (de Lima et al. 2013). The drier northeast of the island is subject to frequent fires, and has many deciduous tree species (Leventis and Olmos 2009); most of which has been extensively cleared for agriculture and turned into savannah-type habitat (Oliveira 2002, Carvalho et al. 2004). These dry coastal lowland forests have suffered the most, being largely cleared for sugar cane starting in the 16th century (Tenreiro 1961). During the 19th and 20th century, large extents of intensive cocoa and coffee shade plantations, known as “roças” (Oliveira 1993; Frynas 2003) led to deforestation up to 1200 m. Subsequent agricultural abandonment driven by falls in production and global prices of cocoa and coffee led to significant regeneration (Tenreiro 1961; Oliveira 2002; Carvalho et al. 2004). The resulting secondary forests, have lower canopy, higher proportion of introduced species and less biomass (Exell 1944).

The biodiversity of São Tomé is unique and fascinating to many researchers around the world. It is extremely rich in endemic species, with its forests having been identified as the third most important for the conservation of forest bird species worldwide (Buchanan et al. 2011). The endemics are strongly dependent on the native forest (Rocha 2008; de Lima 2012; Soares 2017), most of which is now included in the 295 km2 Obô Natural Park (STONP) (Dallimer et al. 2009; Soares 2017). This park was created in 2006 to protect native fauna and flora, as well as their habitats (Direcção Geral do Ambiente 2006). A buffer zone was also envisioned but never officialized. The STONP action and management plans have been created and revised (Albuquerque et al. 2008; Albuquerque et al. 2014), but implementation remains weak (de Lima et al. 2015).

The avifauna of São Tomé is particularly remarkable, including at least 17 single-island endemic species, three endemic species shared with other islands in the Gulf of Guinea, and eight endemic subspecies of widespread species (Jones & Tye 2006). These high levels of endemism have led to São Tomé having multiple Important Bird Areas and to be one of the smallest high priority Endemic Bird Area at a global level (Dallimer & King 2008). Twenty out of the 50 terrestrial bird species that breed on the island are endemic, nine of which are globally threatened, including three Critically Endangered, one Endangered, and five Vulnerable, plus two Near Threatened (IUCN 2017). Notably the most threatened species are those that are most dependent on native forest (Dallimer et al. 2009). The right amount of isolation allowed many species to evolve in environments distinct from those found in the mainland (Miller et al. 2012). São Tomé Island also holds many bird species that exhibit several island syndromes, such as dwarfism and giantism. In islands, small species tend to get bigger, while large species tend to get smaller (Meiri et al. 2006). The Giant Sunbird Dreptes thomensis, the Giant Weaver Ploceus grandis, and the São Tomé Grosbeak Neospiza concolor are all the largest within the respective genera, while the São Tomé Speirops Zosterops lugubris and the São Tomé Thrush Turdus olivaceofuscus are the largest African representatives within the respective genera. On the other hand, the largest endemic bird species in São Tomé, the Dwarf Ibis Bostrychia bocagei is smaller than the mainland counterparts (Melo 2006; Melo et al. 2017).

Previous studies on São Tomé have emphasized that a significant number of island endemics also use human-modified habitats, such as secondary forests and shade plantations (Rocha 2008; Dallimer et al. 2012; de Lima et al. 2012; Soares 2017). Still, there are limits to how much change endemic species can withstand and so, although there is still much forest left on São Tomé, the fast growing human population and resulting urbanization will lead to an increase in natural resource use. These critical changes are likely to lead to a critical decrease of population size or even extinction of most endemic birds (Dallimer et al. 2009). Enforcing strong hunting regulations, like establishing hunting periods and specific quotas for abundant species, is a key conservation need. As is raising awareness, since most São Tomé hunters and game consumers are not aware of the difference between endemic, native or exotic species, or the conservation problems resulting from overhunting and logging (Carvalho et al. 2016). One of the best strategies to avoid this particular threat is to involve the population in conservation efforts, namely by sharing their knowledge and putting in use to support conservation.

This thesis has two main goals, relating to São Tomé common bird species. In the first chapter, we study the coloration and biometrics to determine age and sex. In the second chapter, we assess breeding and moulting seasonality, relating it to species traits and local environmental conditions. Improving our knowledge on these aspects of bird reproduction is key to develop effective conservation strategies.

CHAPTER 1: Coloration and morphology of endemic birds for sex and age identification

Abstract: Determining the sex and age of individuals in natural populations is important for ecological studies and for conservation. However, for many bird species, sex and age are difficult to determine. Island environments offer unique opportunities to study the evolution of sexual dimorphism because insular populations frequently face different predation pressures, feeding conditions, intraspecific and interspecific competition than continental populations. The main goal of this chapter is to gain a better knowledge of the sexual dimorphism of São Tomé bird species. Field measurements, photographs and the molecular sexing of 1046 individuals, allowed identifying sex and age related characteristics for the 12 species most often captured in mistnets. Eight of these were sexually-dimorphic, while the remaining four could not be sexed based on observable characters. Using generalized-linear models based on biometrics to develop morphometric-based discriminant functions allowed a better sex distinction of three species. The incorporation of information on altitude and habitat type further allowed distinguishing male and female São Tomé Weavers Ploceus sanctithomae, while improving the ability to distinguish the sex of most other species. Coloration was crucial to identify age in most species, and contributed to sex adults in six species of which two, only adult males could be identified with a high degree of confidence. This study greatly improved our ability to sex the endemic birds of São Tomé in the field, a crucial information both for fundamental research on ecology and evolution and for the conservation of this unique community.

Keywords: conservation, discriminant functions, endemism, morphometrics, sex identification

INTRODUCTION

Sexual dimorphism, where sexes have distinct phenotypes, is a common and often substantial form of intraspecific phenotypic differentiation, such as in colour, shape, size, structure or behavior. It can occur in any group of living beings, from protists to plants and animals (Punzalan & Hosken 2010). The differences in coloration found between sexes can be explained by the different climates, the presence of predators and of parasites (Covas 2011). When intra-specific competition for resources is high in environments where interspecific competition is small sexes tend to diverge morphologically for ecological reasons: unequal-sized males and females can exploit different sizes of food leading to a decrease in competition for resources. In some plants, the dissimilarities are only functional, not competitive (McGee & Wainwright 2013). Many birds show some dimorphism in terms of coloration, usually the female being cryptically coloured to remain hidden on the nest while the more-colourful male uses it to demonstrate territorial and courtship behaviours (McGee & Wainwright 2013). The function of sexual dimorphism, in many cases, is related to the competition of individuals for access to reproduction, using such characters to attract or fight for a partner (Hamilton 1961). The differences may be extreme, as in the variety seen in the exotic plumages and colours of the male species or in the adaptations for protection (Johnsen et al. 2003).

Islands have a smaller number of species compared with the mainland regions and have been studied for their capacity to offer insights into evolutionary processes (Grant 1998; Whittaker & Palacios 2007; Roulin & Salamin 2010). A reduction in interspecific competition, predation and other factors might allow island organisms to achieve optimal morphological adaptation resulting in a different degree of sexual dimorphism in comparison to the more complex mainland assemblages (Greenberg & Danner 2013). When a taxon colonizes a new habitat it starts the process of adaptation

5 to this new environment. In the case of islands, which are often characterized by reduced interspecific competition, and a different distribution of resources, species tend to have wider ecological niches, resulting in: (A) sub-niches being occupied by all individuals; (B) intraspecific sub-niches linked to sex; or (C) specific sub-niches unrelated to sex (Greenberg & Olsen 2010). These patterns in islands are associated with rapid morphological divergence, often involving strong morphological divergence from their continental relatives, sometimes resulting in large radiations (Pratt 2005; Grant 2008; Alström et al. 2015). In birds, some genera like Terpsiphone and Zosterops, have a high propensity to colonize islands and to speciate rapidly once there (Moyle et al. 2009; Fabre et al. 2012). Island environments can lead to changes in morphological divergence as a result of changes in social interactions (Robinson-Wolrath & Owens 2003). Mating and parental care strategies may change after island colonization (Robinson-Wolrath & Owens 2003; Covas 2011). In birds it is frequently believed that insular taxa develop dull coloration and are less sexually dichromatic likely due to a decrease in the intensity of sexual selection but supporting evidence remains limited (Doutrelant et al. 2016). This indicates that we have opposite predictions in islands depending on whether the ecological or sexual factors play a dominant role.

To date there is no generally recognized method to record age in birds. Juveniles tend to have their first complete moult as soon as they leave the nest (Demongin 2016). However, they still have some distinctive signs easily to recognize like feathers that tend to be weaker and looser than the adults; growth bars on tail and wing feathers; the shape of the tail feathers that tend to be narrower and more pointed than the adults. Also iris and bill coloration can vary with age or sex, generally duller and darker in juveniles than in adults and in adult females when compared to adult males (Stevensson 1992, Demongin 2016). Often gape flanges are also used to identify juveniles but it should be noted that some species have flanges as adults (Stevensson 1992). The adult plumage is definitely acquired and does not change the appearance with age and successive moults, except between seasons e.g. breeding (Demongin 2016).

Determining the sex and age of individuals in natural populations is a useful tool for studying population dynamics, population structure, habitat use, behavior and mating systems, species vulnerability to extinction, distribution shifts and therefore for making appropriate management decisions, both at the habitat and species level (Shaw et al. 2003); Schwartz 2012). Accurate and easy methods to determine the sex of individuals are thus valuable for ornithological studies. Unfortunately, for many bird species it is difficult to determine sex based solely on morphometrics and coloration (Calabuig et al. 2011). The sex of birds is commonly determined by laparoscopy, cloacal examination, genetic analyses and morphometrics. All of these techniques require capturing birds and the use of procedures that compromise the wellbeing of birds. Non-invasive sexing can be inferred by differences in coloration, morphology and behavior, but are overall less accurate than more invasive techniques (Volodin et al. 2015). Molecular sexing is one of the simplest and most used DNA-based techniques applied to wild birds. For obtaining genetic material, the most common sampling method for sexing is the removal of blood samples, because blood is fairly easy to take from captured individuals and have a large amount of DNA (Harvey et al. 2006). Feather samples have been used less often for molecular sexing, but they too represent an appealing sampling method, because removing a single feather is one of the least invasive methods of obtaining a genetic sample (Harvey et al. 2006). Genetically-based sexing is very reliable, but it is relatively expensive and time-consuming. Additionally, incorrect settings of genetic analyses may, to some extent, decrease the reliability of this method (Volodin et al. 2015).

Morphometric-based sexing depends on measuring variances in body size between sexes. It requires minimal equipment and staff training, and provides immediate results. However, its reliability

6 can be limited and it decreases when sex dimorphism is reduced (Volodin et al. 2015). Discriminant functions analyses (DFA) are a popular statistical tool because it can classify individuals of unknown origin into groups, classes or categories of the same type, using a discriminant function (DF) generated from a data set composed of individuals of known origin (White & Ruttenberg 2007). The use of DFA using morphological measurements has proved useful to determine sexes (Berkunsky et al. 2009). However, the skill of the researcher for sex estimation using DFA plays an important role (Moore 2013). DFA may incorporate not only characteristics of the birds, but also the locations where they are captured and/or the time of year. Molecular sexing is a very good complement to DFA in species with unclear sexual dimorphism, since the human ability to always take the same measurements for some morphometric-based sexing plays a big role in the percentage of human error (Calabuig et al. 2011), although mass is clearly less dependent on who is measuring than other measures.

The island of São Tomé is one of the 218 Endemic Bird Areas identified worldwide (Dallimer et al. 2009). It is home to twenty endemic bird species, of which three are considered Critically Endangered, one Endangered, six Vulnerable, two Near Threatened and eight Low Concern (IUCN 2017). The main goal of this chapter is to gain a better knowledge of the sexual dimorphism of São Tomé common bird species. Specifically, we intend to:

• Identify characteristics that allow identifying sex and age;

• Use morphometrics to create functions that allow discriminating sexes;

• Assess if habitat and altitude can improve the ability to distinguish sexes.

We will thus verify and complement the knowledge on the sexual dimorphism of this endemic-rich community (HBW Alive 2017; Naurois 1994; Jones & Tye 2006).

METHODS

Study area

The 857 km2 oceanic island of São Tomé (0º25’N-0º01’S, 6º28’E-6º45’E) is the largest island of the Democratic Republic of São Tomé and Príncipe. Located in the Gulf of Guinea, it is part of the Cameroon line (Fig. 1.1). It lies 255 km west of mainland of Africa (Gabon) and 150 km south- southwest of Príncipe Island (Peet & Atkinson 1994). Its location has allowed the development of high levels of endemism, being far enough to be isolated, while at the same time allowing a fairly high number of potential colonizers (Melo 2007; Melo 2012). The highest elevation point is “Pico de São Tomé” at 2,024 m a.s.l.. The mountainous center of the island halts the dominant southwest winds, originating a very strong rainfall gradient across the island. Annual precipitation varies from less than 600 mm in the northeast to over 7,000 in the southwest. Despite its proximity to the equator, São Tomé has two clear main seasons: the dry season, between June and August, called “gravana”, and a rainy season, from September to May. There is also the “gravanito”, a smaller dry season between December and January (Leventis & Olmos 2009). The average temperature is regularly constant throughout the year, oscillating between 18°C and 33°C across the island (Coelho 2016).

São Tomé was originally covered in forest, except for tiny areas of rocky outcrops (Peet & Atkinson 1994). There are three key distinct types of forest: Lowland forest, which extends from the

7 sea level up to 800 m, and most of which is nowadays cultivated or savanna in the north of the island where rainfall is very low; Montane forest, which extends from 800 m to 1,400 m, composed by a dense canopy and by tall trees covered by epiphytes, and currently threatened by agricultural development; and Mist forest, extending from 1,400 m to 2.024 m (Exell 1944) it has an open canopy, the lowest temperatures and is of difficult access, which has kept it protected from most human activities (de Lima et al. 2013).

Figure 1.1- Map of São Tomé Island in relation to mainland Africa (Small inset on the bottom right). The sampling locations are shown by the orange dots. The initial 37 sampling locations were grouped in 23 map locations to allow visualization, since some of them were in close proximity of each other.

Data collection

We compiled the bird ringing data that has been collected in São Tomé Island since 2002 by Martim Melo and colleagues. These were collected throughout the year, in 30 sampling locations across the island. We collected additional data between mid-January and the end of March 2017, respecting the same locations used in the years before. We used vertical understory mist-nets to capture the birds (De Beer et al. 2001). The location and number of nets was decided on the spot, to maximize diversity and number of birds captured, while taking into consideration our capacity to process them and external factors, such as weather. Whenever possible, nets were opened just before dawn and kept open until we could make sure that the birds could be safely released on the same day. Each location was sampled for three days, which has be shown to be the ideal period to minimize bird mist-net avoidance ((Marques et al. 2013), tested locally).

All birds were identified to species level, and all first captures were ringed. Whenever possible, sex and age were determined in the field, even if tentatively, using size, plumage, moult, breeding marks and any other physical traits such as iris and bare parts colour. To determine sex-specific morphometrics we measured: the length of the wing (distance between the carpal joint and the tip of the longest primary, measured on the closed wing), the length of the tail to the nearest half-millimeter, using a ruler, the length of the tarsus measured to the 10th of a millimeter using a digital caliper, and the weight (measured using a scale or a dynamometer, appropriate to the weight of the bird).

Some birds were sampled for genetic material, by collecting blood samples or two or three plucked feathers. The blood samples were obtained from the brachial vein in the wing and we collected approximately one or two drops of blood in capillary tubes via brachial venipuncture with a sterile hypodermic needle. Blood samples were immediately transferred to tubes containing 0.5 ml of blood lysis buffer. Feather samples were obtained from the individuals by pulling two or three feathers one at a time from the breast of each bird. Feather samples were then kept in individual paper envelopes. The blood and feather samples were labelled for molecular sexing, and subsequent association with field measurements. Molecular sexing followed the protocol of Griffiths et al. (1998). We combined non-invasive techniques with one invasive technique (DNA collection) to improve our ability to correctly sex individuals of each study species and therefore reduce the error of each technique and to look for sets of traits in discriminant functions analyses (DFA) capable of identifying sex for those species whose sexual dimorphism is small or still poorly understood e.g. confusion with age as in São Tomé Weaver or with moult patterns as in São Tomé Paradise-flycatcher Terpsiphone atrochalybeia.

Whenever possible we took photos from multiple perspectives of the birds in the hand, and took notes on individual morphologic characteristics, such as feather, eye and beak coloration and moult scores, for subsequent association with sex and age. These photos were organized and categorized by species, and then by sex and if possible age. The files were named using capture date and ring code, to make it easy to link with the information on the database. Our records, including those obtained from molecular sexing, were added to the São Tomé bird ringing database, which was then scanned for errors, and standardized.

Data analysis

To ensure we could perform robust statistical analyses, we selected for subsequent analyses only the 12 species that had over 100 records from the initial 39 species. We discuss sexual dimorphism for all these 12 species, but focus on those for which the sexual dimorphism is unknown or not clear. To assess sexual dimorphism we used boxplots, scatterplots and Kruskal-Wallis tests to compare field measurements (weight, except possible females with egg; wing, tail and tarsus length) of each sex (as determined by molecular analyses) for each species. We took into account the possibility of abrasion of the flight-feathers and tail while measuring birds on the field.

Information on morphometric and other external characteristics that allow sex and age identification was compiled for each São Tomé common bird species. The photographs of molecularly-sexed individuals were checked to identify additional characteristics that could allow sex determination. Finally, we used generalized linear models (GLM) with binomial error distribution to create sex discriminant functions based on morphometrics. Habitat were categorized as forest or plantation and altitude as montane or lowland, based on whether they were located above or below 800 m a.s.l., respectively. Habitat and altitude were subsequently added to the GLMs, to evaluate if they increased the ability to discriminate sexes.

RESULTS

We obtained 6125 ringing records, belonging to 39 species. These were collected since 2002, and include 761 new records from 2017. There were 12 species that had over 100 records, henceforth referred to as São Tomé common bird species (Table 1.1). For some species we already had an idea of

9 the sexual dimorphism and for others we were almost certain from the field observations but to test this, 1046 individuals were molecularly sexed from blood and feather samples. We photographed 759 individuals, 258 of which were sexed using molecular techniques, which allowed confirming external morphological characters associated with sex.

Table 1.1- List of São Tomé common birds, including the number of valid records (n), the number of individuals that were sexed using molecular techniques (n sexed – percentage of the “n” shown in parenthesis), the number of sexed individuals that also had photos (n photos – percentage of “n sexed” shown in parenthesis) and the knowledge of sexual dimorphism prior to this study in colour, size our both; those that do not appear to have any sex dimorphism (not known) and others that seem to have almost certainly sex dimorphism especially in morphometrics (in bolt) and others in which there were doubts for different reasons (?). The species are ordered alphabetically, according to the scientific name.

English name Scientific name n n sexed n photos prior Newton Sunbird Anabathmis newtonii 662 66 (10%) 17 (26%) colour Lemon Dove Columba (larvata) simplex 139 52 (38%) 18 (35%) not known Príncipe Seedeater Crithagra rufobrunnea 462 160 (35%) 25 (16%) not known Giant Sunbird Dreptes thomensis 136 27 (20%) 10 (37%) size Common Waxbill Estrilda astrild 254 55 (22%) 6 (11%) colour colour & 126 61 (48%) 18 (30%) Giant Weaver Ploceus grandis size São Tomé Weaver Ploceus sanctithomae 347 149 (43%) 79 (53%) ? São Tomé Prinia Prinia molleri 340 62 (18%) 10 (16%) ? São Tomé Paradise- 485 53 (11%) 16 (30%) colour flycatcher Terpsiphone atrochalybeia São Tomé Thrush Turdus olivaceofuscus 882 88 (10%) 12 (14%) not known São Tomé White- 109 46 (42%) 2 (4%) not known eye Zosterops feae Black-capped 1727 222 (13%) 45 (20%) not known Speirops Zosterops lugubris

Morphometrics and sex

Except for the São Tomé White-eye species, there were significant morphometric differences between sexes for all species (Fig. 1.2 & Fig. S1). The Newton Sunbird, the Giant Sunbird, the Giant Weaver and the São Tomé Weaver showed significant differences between males and females for all measurements (Fig. S1). When there were differences, males were larger than females, except for the weight of the São Tomé Thrush. Out of the four measurements, wing length differed between sexes in 10 out of 12 species, whereas weight and tarsus length differed only in six species.

Despite the large number of significant differences between sexes, most morphometrics revealed a wide overlap, making it difficult to use them as diagnostic characteristics. The exceptions to this were the Giant Sunbird and the Giant Weaver. On the other hand, despite having some sexual dimorphism in coloration, sexes of the São Tomé Paradise-flycatcher are very difficult to distinguish using morphometrics.

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Distinguishing sex and age: Individual species accounts

Newton Sunbird Anabathmis newtoni. Adult has a clear sexual dimorphism (Fig. 1.3): the male has a metallic blue throat and sunny yellow breast and belly, while the female has as a brownish throat and dull yellow underparts. Males are larger than females for all morphometric traits (Table 1.2). The coloration pattern of the juvenile is identical to that of the females (Fig. 1.3), and can only be distinguished by the overall weaker quality of the feathers or other general characteristics of juvenile birds, such as a bright, well-developed, gape.

Figure 1.3- Photos of Newton Sunbird. From left to right: adult male, adult female and juvenile.

Lemon Dove Columba larvata simplex. Sexual dimorphism is present but much reduced in adults: the eye ring (bare skin) is red in males and grey in females; wings are often longer in males (Table 1.2). The juveniles are much browner, less iridescent, than adults (Fig. 1.4), with the wing coverts being fringed with golden orange bars; the iris is dark reddish-brown and the bill is light pinkish grey, compared to the red iris and dark bill of the adults.

Figure 1.4 - Photos of Lemon Dove. From left to right: adult male, adult female and fledgling.

Príncipe Seedeater Crithagra rufobrunnea. No sexual dimorphism was detected (Fig. 1.5). The length of the wings is the only measurement that can distinguish male and female, but only for extreme values (Table 1.2). Juveniles can only be distinguished by the overall weaker quality of the feathers and other general characteristics of juvenile birds. Although we suspect that there is a possible difference in the coloration of the iris of adults and juveniles, this was difficult to demonstrate due to the influence of light conditions in our assessment of colour.

Figure 1.5 - Photos of Principe Seedeater. From left to right: adult male, adult female.

Giant Sunbird Dreptes thomensis. Strong sexual dimorphism in morphometrics but not on colour: females are smaller than males with no overlap in any of the measurements, except in a few occasions on the tail (Fig. 1.2, Table 1.2). The bill of females is visibly shorter than that of males, in proportion to body size. The coloration pattern of the juvenile is identical to that of adults (Fig. 1.6) and can only be distinguished by the overall weaker quality of the feathers or other general characteristics of juvenile birds, such as a protruding gape.

Figure 1.6 - Photos of Giant Sunbird. From left to right: adult male and female.

Common Waxbil Estrilda astrild. The sexes of adult Common Waxbill can be identified by the undertail coverts, which are dark brown to completely black in the males and paler and visibly barred in the females (Fig.1.7). Males also have a pinkish-red flush along the belly, which is paler on females (Fig. 1.7). For extreme values, the sexes can be distinguished based on tail length (Table 1.2). Recently fledged juveniles are easily distinguished by the black bill and white gape flange which turns red with hints of brown in older juveniles; the bill of the adults is reddish orange and has a very small dark gape (Fig.1.7).

Figure 1.7 - Photos of Common Waxbill. From left to right: adult male, adult female and juvenile. The female of this photo have dirt on its bill.

Giant Weaver Ploceus grandis. Species with a clear sexual dimorphism in adults in all morphometric characters (Table 1.2) and colouration. The head of males is black, down to the chin and throat, blending into chestnut-brown on the rear of the crown, neck and breast; the mantle and upper back are greenish yellow to olive, ending with black feathers with yellow tips, which continue to the wings; the primary feathers are all black; the belly and flanks are bright sunny yellow, as is the iris; the uppertail coverts are dark yellow, the tail is dark olive brown, and the bill is black and massive (Fig.1.8). The females have a brown head with a pale throat; the bill is also strong and completely black; the iris is a dull yellow; the breast is dark yellow to orange mottled with light brown, and blending into a brownish flank; the belly is white bending with the flanks; the wings have dark feathers with yellow tips; the mantle to the upper back is dark yellow to brownish (Fig. 1.8). The juveniles can be told apart from the females by: a non-black bill (dark brown upper mandible and pinkish brown lower mandible) with yellow and enlarged gape flanges in very young birds; a dark brown iris that becomes lighter with age; light flanks and a white belly; a paler breast with a yellowish brown colour; lighter olive brown head and upper back. The wings of the juveniles are similar to those of female, but are more dark brown than black.

Figure 1.8 - Photos of Giant Weaver. From left to right: adult male, adult female and juvenile.

São Tomé Weaver Ploceus sanctithomae has no clear sexual dimorphism. Individuals of this species show a wide range of plumage colour intensity: from well-marked individuals, with a black crown and bright orange plumage, to dull individuals with a very faint crown. The locals associate these extremes with male and female plumages and so do some authorities (Craig 2017). Using molecular sexing, this study demonstrates that there is no colour dimorphism in the adults. The colourful individuals are adults, the dull ones juveniles. In the youngest juveniles there is no distinguishable crown, the bill is pink, since the upper mandible has no light brown (Fig. 1.9). The gradation in colour intensity in the adults is more likely related to wear, as suggested by Christy and Clarke (1998). In the adults, morphometric traits can distinguish the large males from the small females in every trait (Table 1.2).

Figure 1.9 – Photos of São Tomé Weaver. From left to right: adult male, adult female and juvenile. 14

São Tomé Prinia Prinia molleri. Species without clear sexual dimorphism. The males have a chestnut to orange face and a rufous-brown crown merging into grey on nape, a pattern that is more faded on females (Fig. 1.10). Males also tend to have a longer tail, which allows sexing for extreme values (Table 1.2). The coloration pattern of the juvenile is almost identical to that of adult females but with a yellow wash in the throat and the breast (Fig. 1.10), and can be distinguished by the overall weaker quality of the feathers; the iris is greyer than in the adults which is warm brown. The inner lower mandible is more yellow pink in juveniles and pink in adults.

Figure 1.10 - Photos of São Tomé Prinia. From left to right: adult male, adult female and juvenile.

São Tomé Paradise Flycatcher Terpsiphone atrochalybeia. The adult São Tomé Paradise- flycatcher has a clear sexual dimorphism in plumage colour. The adult males have an entirely dark- blue glossy dark-blue plumage; the eye has a light-blue featherless eye ring; with two central streamers that can reach up to 11.5 cm beyond the remaining tail feathers (Fig.1.11). The females have a rusty red mantle and back; the nape, lores and throat are light grey; they have a glossy/ iridiscent blue crown; the light-blue featherless eye ring is less developed and duller than that of males; the breast, belly and flanks are pale grey; the primaries and secondaries are dark brown; the tail is rusty- red with a dark patch at the end (Fig. 1.11). The juveniles are difficult to sex as they are very similar to adult females. Nevertheless, they are duller, especially in the rusty-red plumage and in the sky-blue eye ring. Fledglings have grey faces and small dull eye rings, and the bill is yellowish with a black tip (Fig.1.11). It appears that juvenile males may undergo more than one moult before reaching the full black adult plumage. Such putative immatures or young adult males have a few dark-blue iridescent feathers scattered across a female-looking plumage pattern. The sexes can be distinguished when the induviduals present extreme values of weight and tarsus length (Table 1.2).

Figure 1.11 - Photos of São Tomé Paradise-flycatcher. From left to right: adult male, adult female and juvenile. 15

São Tomé Thrush Turdus olivaceofuscus. Species with reduced sexual dimorphism. Adult males have a red iris, whereas the females have a chocolate brown iris (Fig. 1.12 & Fig. S2). Morphometric characters allow distinguishing males and females at extreme values of weight, tail length and especially wing length (Table 1.2). The coloration pattern of the juvenile is identical to that of adults, although they tend to have more white feathers in their chest and depending on the age the iris can be brownish-grey, brown or brownish-red. Young juveniles have the wing coverts tipped with light rusty- brown as typical of the Turdus genus.

Figure 1.12 - Photos of São Tomé Thrush. From left to right: adult male, adult female and juvenile.

São Tomé White-eye Zosterops feae. Species with no sexual dimorphism (Table 1.2). Juveniles are indistinguishable from adults (Fig. 1.13), except for the overall weaker quality of the feathers and, depending on the age of the juvenile, they can have some of their orbital feathers still growing. The tip of the bill is generally lighter grey in juveniles.

Figure 1.13 - Photos of São Tomé white-eye. From left to right: male and female.

Black-capped Speirops Zosterops lugubris. Species with very little sexual dimorphism. Sexes can only be distinguished using morphometric characters in extreme cases (Table 1.2). Juveniles’ coloration pattern is identical to that of adults (Fig. 1.14). Juveniles can be distinguished by the same characteristics as the juveniles of São Tomé White-eye.

Figure 1.14 - Photos of Black-capped Speirops. From left to right: adult male, adult female and juvenile.

Table 1.2 – Measurements of São Tomé common bird species. These values refer to all individuals that were measured, and then sexed using molecular techniques. The values show the range of measurements that fall within a 95% confidence interval, assuming a normal distribution, and additional maximum and minimum values in parenthesis when there are outliers that fall outside that range. The species are ordered alphabetically, according to the scientific name and are indicated by a combination of the three first letters of the generic name and the first three letters of the specific name (see Table 1.1). The letter “M” represents the males and the letter “F” the females.

Tail Species Sex Mass (g) Wing length (cm) Tarsus length (cm) length (cm) Ananew M 6,5 - 8.0 (48) 50 - 57 31 - 45 (18.3) 18.5 - 20.4 (n=66) F 5.0 - 7.0 47 - 51 30 - 38 (16.0) 17.9 - 19.4 Collar M (90) 135 - 235 150 -165 (169) 82 - 109 37.8 - 44.6 (n=52) F (110) 130 - 210 142 - 159 (160) 83 - 100 37.5-42.4 Criruf M (16) 20.5 - 26.5 (27) 78 - 87 50 - 62 (20.3) 22.8 - 44.6 (n=160) F 19 - 27 77 - 86.5 48 - 62 22.9 - 25.8 (20.5) Dertho M 24.2 - 30.5 87 - 94 75 - 100 31.7 - 34.2 (n=27) F 24 - 18 81 - 85 65 - 82 29.5 - 30.9 Estast M 6.9 - 8.9 44.5 - 48.5 43 - 50 14.9 - 17.9 (n=55) F 6.5 - 9.0 43 - 48.5 40 - 47 15.1 - 17.3 Plogra M 63 - 78 (102) 107 - 116 (119) (65) 67 - 75 (30.2) 32.6 - 37.5 (n=61) F 48 - 60 101 - 108 63 - 72 (27.5) 31.2 - 33.5 Plosan M (19.5) 20.4 - 25 (27.3) 74 - 82 44 - 54 (22.8) 25.3 - 28.6 (21) (n=149) F (13.4) 15.4 - 24.5 68 - 77 41 - 53 23.35 - 27 Primol M 8.2 - 10.9 (11.3) 50 - 58 (65) 67 - 83 (22.1) 22.5 - 25.3 (n=62) F 8.0 - 11.5 48 - 56 50 - 81 21.6 - 25.1 Teratr M 12 - 15.9 75 - 81 (84) 81 - 98* (20.4) 22.6 - 23.8 (n=53) F (11) 11.4 - 13.8 74 - 82 (83) 81 - 90 21.6 - 23.7 Turoli M 67.5 - 94 (104) 123 - 141 89 - 108 (39.8) 41.8 - 48.21 (n=88) F 75.3 - 100 (116) 121 - 132 86 - 107 40 - 46.6 Zosfea M 6.7 - 9.2 54 - 56 (57) 36 - 42 (17) 17,6 - 20 (n=46) F 7.2 - 8.4 (8.9) (52) 52.5 - 56 36 - 39 (16.6) 17.8- 20.2 Zoslug M 13.9 - 20.4 (69.5) 70 - 77 46 - 60 (22.2) 23.1 - 26.7 (n=222) F 14.1 - 19.8 68 - 76 46 - 62 (21.4) 23.4 - 26.5 (26.7)

In summary (Table 1.3), the combination of external morphological characters allows identifying with certainty the juveniles of Lemon Dove, Common Waxbill and Giant Weaver, and most times those of São Tomé Prinia, São Tomé Thrush and São Tomé Paradise-flycatcher. It also allows sexing the adults of Newton Sunbird, Giant Sunbird, Giant Weaver, São Tomé Thrush and São Tomé Paradise-flycatcher, and sometimes the juveniles of Newton Sunbird, São Tomé Thrush and São Tomé Paradise-flycatcher.

Table 1.3 – Sex and age distinctiveness of São Tomé common bird species, based on morphometrics and coloration. Alphanumeric code reveals when sex and age can be distinguished: 0 - rarely; 1 – sometimes; 2 – most times; 3 – always; N – never; M - only adult males; \J – always, except for juveniles. The species are ordered alphabetically, according to the scientific name.

Sex

Specie/Distinctive features Age Mass Wing Tail Tarsus Coloration Newton Sunbird 2 2 2 2 2 M

Lemon Dove 3 0 1 0 1 \J Principe Seedeater 0 0 1 0 0 N Giant Sunbird 0 3 3 2 3 N Common Waxbill 3 0 1 1 1 \J Giant Weaver 3 3 3 1 2 \J

São Tomé Weaver 1 2 2 1 1 N São Tomé Prinia 2 0 1 1 1 N São Tomé Paradise-flycatcher 2 1 0 1 1 M São Tomé Thrush 2 1 1 0 1 \J São Tomé white-eye 0 0 0 0 0 N

Black-capped Speirops 0 0 1 0 0 N

Morphometric-based sex discriminant functions

The development of sex discriminant functions using morphometric-based GLMs allowed sexing the Newton and the Giant sunbirds and the Giant Weaver with full confidence (Table 1.4). The ability of these functions to discriminate the sex of the São Tomé Weaver was also very high. As expected from the almost complete overlap in all morphometric traits, the São Tomé White-eye and Black- capped Speirops were mostly indistinguishable.

Table 1.4 - Sex discriminant functions using morphometric-based GLMs for the São Tomé common bird species. The equations were used to calculate the discriminant scores (DS) for each species indicated by a combination of the three first letters of the generic name and the first three letters of the specific name. The sample size (N) for males (M) and females (F) is shown, as is the proportion of correctly classified (true positives) males, females and sexes (T). The coefficient of determination (R2) gives an idea of the proportion of variability that is explained by the model, ranging from zero, when it is not at all explained to one, when it is fully explained by the model. The species are ordered alphabetically, according to the scientific name and are indicated by a combination of the three first letters of the generic name and the first three letters of the specific name.

N True positives Sp Discriminant function R2 M F M F T DS=(-16.68 x Mass)+(-48.83 x Wing)+(2.41 x Ananew 33 14 100 100 100 1 Tail)+(-36.54 x Tarsus) DS=(-0.01 x Mass)+(-0.22 x Wing)+(0.03 x Tail)+(- Collar 14 16 57.1 87.5 73.3 0.22 0.11 x Tarsus) DS=( 0.23 x Mass)+(-0.53 x Wing)+(-0.004 x Criruf 82 50 82.9 54 72 0.17 Tail)+(0.05 x Tarsus) DS=(-6.10 x Mass)+(-5.71 x Wing)+(2.09 x Tail)+(- Dretho 9 10 100 100 100 1 8.76 x Tarsus) DS=(0.92 x Mass)+(1.21 x Wing)+(-3.62 x Tail)+(- Estast 7 11 71.4 81.8 77.8 0.63 0.87 x Tarsus) DS=(-4.68 x Mass)+(-9.81 x Wing)+(2.81 x Tail)+(- Plogra 22 22 100 100 100 1 2.98 x Tarsus) DS=(-2.44 x Mass)+(-0.51 x Wing)+(-0.24 x Tail)+(- Plosan 80 47 100 95.7 98.4 0.92 1.10 x Tarsus) DS=(0.25 x Mass)+(-0.20 x Wing)+(-0.28 x Tail)+(- Primol 19 20 84.2 80 82.1 0.45 1.07 x Tarsus) DS=(-0.64 x Mass)+(-0.32 x Wing)+(-0.14 x Tail)+(- Teratr 15 15 73.3 80 76.7 0.23 0.18 x Tarsus) DS=(0.22 x Mass)+(-0.46 x Wing)+(-0.08 x Tail)+(- Turoli 33 18 87.9 77.8 84.3 0.46 0.54 x Tarsus) DS=(-0.31 x Mass)+(0.10 x Wing)+(-0.33 x Tail)+(- Zosfea 25 17 88 11.8 57.1 0.03 0.11 x Tarsus) DS=(0.012 x Mass)+(-0.31 x Wing)+(-0.03 x Tail)+(- Zoslug 91 82 68.1 48.8 59 0.05 0.10 x Tarsus)

The inclusion of habitat and altitude in the GLMs improved considerably the ability to discriminate sexes for all species (Table 1.5). Adding both environmental variables allowed identifying the sexes of São Tomé Weaver with full confidence, of the São Tomé Thrush in over 90% of the cases, of the Lemon Dove in over 80% of the cases, and of both the Principe Seedeater and the São Tomé Paradise- flycatcher in over 70% of the cases. The inclusion of altitude alone allowed the correct sexing of São Tomé Prinia and São Tomé Thrush in over 90% of the cases and of the Common Waxbill in over 80% of the cases. Habitat alone had the lowest impact in discriminatory power for all species except for São Tomé White-eye and the Black-capped Speirops that had the percentage of confidence over 60% of the cases.

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DISCUSSION

The use of field measurements, photographs and molecular sexing allowed assessing sexual dimorphism in 11 common endemic bird species from São Tomé and the non-endemic Common Waxbill. Only Newton Sunbird, Giant Sunbird, Giant Weaver and São Tomé Paradise-flycatcher had clear sexual dimorphism. Distinguishing the sex of Newton Sunbird individuals was possible using morphometric-based discriminant functions. The incorporation of environmental variables allowed distinguishing male and female São Tomé Weavers, while improving the ability to distinguish sexes in most other species. The São Tomé White-eye and the Black-capped Speirops do not have any sexual dimorphism. The ability to distinguish between the sexes increased considerably when considering altitude and habitat, which suggests that there is differentiation at very small scales.

Age determination

Colouration

Coloration allowed to directly inferr the age of birds for three species, the Lemon Dove, the Giant Weaver and Common Waxbill. In the other cases, juveniles could only be identified when any of the general characteristics of juvenile birds were detected. Patterns of plumage and bare part differentiation in the island species corresponded well with those described for their mainland counterparts. The eye-ring and eye colour of the Lemon Dove have not been mentioned in the literature (Naurois 1994; Leventis & Olmos 2009; Baptista et al. 2017) as age-related traits in the mainland populations of Columba larvata (Baptista et al. 2017). The age of the closest continental relative of the Giant Weaver, the Vieillot's Black Weaver (Ploceus nigerrimus) can also be distinguished by the coloration of the bill and plumage. The colour identification traits of the Common Waxbill that we detected are the same on the mainland population (Craig 2017, Payne 2017). Knowing the sex of the adults based on the plumage coloration can sometimes be useful on the identification of the sexes of juveniles.

Coloration also enabled, most times, distinguishing the age of five species, the Newton Sunbird, the São Tomé Weaver, the São Tomé Prinia, the São Tomé Paradise-flycatcher, and the São Tomé Thrush. The coloration allowed us to distinguish, without a doubt, the adult males of Newton Sunbird since juveniles are very alike to the adult females as it happens for its closest mainland relative the Northern Double-collared Sunbird (Cinnyris reichenowi) (Cheke & Mann 2017). São Tomé Prinia juveniles can be distinguished by coloration patterns as most of juvenile birds in the genus (HBW Alive 2017). The juveniles of the São Tomé Weaver and São Tomé Paradise-flycatcher could be distinguished based on the duller coloration and colour of the bill although older juveniles are very similar to adults as it happens for most species of both species genus (HBW Alive 2017). The age of the São Tomé Thrush can be distinguished by their eye colour and juveniles tend to have more white feathers in the chest as previously reported (Collar 2017; Leventis & Olmos 2009). The age of their closest continental relative, African Thrush (Turdus pelios), can also be distinguished based on white chest feathers, although juveniles are similar to adults and there is no reference to eye colour (Collar 2017).

The age of the Principe Seedeater, Giant Sunbird, São Tomé White-eye and the Black-capped Speirops could only be distinguished when general characteristics of juvenile birds were visible. These include: feather weakness, flanges, growth bars (especially on the wings and tail), and pointy flight feathers. In almost all mainland Crithagra seedeaters, the juveniles are very similar to the adult females (HBW Alive 2017). The age of Giant Sunbird unlike most species of its family, cannot be distinguished by coloration (Cheke & Mann 2017).The closest relative of the São Tomé White-eye

21 and of the Black-capped Speirops in the continent, the African Yellow White-eye (Zosterops senegalensis) age can also, only be distinguished by the overall general characteristics of juvenile birds.

In conclusion for three of the twelve species present in our study, coloration helped in the identification of age with a high degree of confidence. For other five species we could identify the age based on coloration most of the times with confidence. The age of the last four species present in our study could only be identified when general characteristics of juvenile birds, like feather weakness, flanges, growth bars (especially on the wings and tail), and pointy flight feathers are present.

Moult

Although the study of moult strategies was not an objective of our work, moult was recorded for some of the birds because it can be a reliable indicator of age at least until they acquire the definitive adult type plumage. Juvenile birds of many species have distinct plumage features (distinct coloration, patterns or structure) that allow the separation from adult type birds that acquire the adult plumage after the first complete moult (Svensson 1992).

Little is known about the moult strategies of the birds of São Tomé. According to the data that were gathered it appears that juveniles of most species become indistinguishable from adults after completing the post-juvenile moult (the first moult after fledging). Therefore, moult is of limited interest to determine age of most São Tomé species. An exception appears to be the São Tomé Paradise-flycatcher where males apparently undergo several moults before acquiring the adult plumage.

The moult strategy of the adult birds in São Tomé seems to be a complete post-breeding moult. Apparently the adult plumage once acquired remains stable throughout the year and there is no partial pre-breeding moult (restricted to body feathers) to acquire a breeding plumage. Our data remain preliminary and further study is required, namely on the timing of the moult and its relation with the breeding season. In São Tomé Island there is less variability in climate conditions, which allows bird to adopt simpler moults strategies in opposition to does that are exposed to not so stable climates which developed more complexed moults as seen in temperate areas (Jenni & Winkler, 1994).

Sexing

Five of our twelve study species have clear sexual dimorphism, three have very little sexual dimorphism and four have no apparent sexual dimorphism. Our study found new information on sexual identifying characteristics for five species that were not mentioned in literature. For the Giant Sunbird we could refine the interval for the size of males; for the Lemon Dove we found that the different colour of the eye-ring was a better predictor of sexes than the colour of their plumage; for the Giant Weaver we found that the body size between sexes do not overlap; for the Prinia we found that males have a longer tail and for the Thrush we found that differences between the iris colour were good predictors of sexes. Comparing to their closest relatives in the continent there were five species that both, relatives and study species, had sexual dimorphism; three that had non or very little sexual dimorphism when compared to their relatives; one that when compared to our sexual dimorphic species didn’t had sexual dimorphism and three that both, relatives and study species, had no sexual dimorphism. However it must be taken into account that in most species there was a lack of information about morphometric measurements in literature.

Sexual dimorphic species

Our data shows that both sunbird species are sexual dimorphic in terms of morphometrics. Newton Sunbird is sexually dimorphic in coloration, but not the Giant Sunbird. This matches what is observed in their closest mainland relatives, the Northern Double-collared Sunbird (Cynnyris reichenowi) and Collared Sunbird (Hedydipna collaris) respectively. It had already been suggested that the males of the Giant sunbird are considerably larger than the females (Cheke & Mann 2017, Naurois 1994, and Leventis & Olmos 2009). Our extensive morphological data set together with molecular sexing have clearly demonstrated this.

The Giant Weaver was one of the study species that showed the highest degree of sexual dimorphism, in terms of morphometrics and colour, similar to what happens to some of its continental relatives, such as Viellots’ Black Weaver (Ploceus nigerrimus) and the Village Weaver (P. cucullatus). Although the colour differences are well known since the description of the species, our study highlighted the large, non-overlapping, differences in body size between the sexes. This new information allows sexing juveniles in the hand. A practical illustration of the size differences is that males and females require different size rings.

The São Tomé paradise-flycatcher is sexually dimorphic. Despite living on islands separated by the African continent, the São Tomé paradise-flycatcher and the Seychelles Paradise-flycatcher (Terpsiphone corvina) may be each-others closest relatives (Bristol et al. 2013), suggesting that these islands were colonised during the same period by the same ancestral stock. These two species share a unique colour sexual dimorphism (with males being fully iridescent black) contrasting with continental Terpsiphone species which are less sexually-dimorphic (Fabre et al. 2012). Still, our data suggested that males undergo several moults (or a long protacted body moult) before acquiring the male plumage. In these instances, sexing can be difficult – although it appears that the presence of scattered black iridescent feathers in an otherwise female plumage only occurs in males.

The Common Waxbill is sexually dimorphic as described for the São Tomé and mainland populations (Svensson 1992; Beaman et a. 1998; Sinclair & Ryan 2004; Payne et al. 2017). The diagnostic features are the colours of the undertail feathers and the intensity of the pinkish-red flush along the belly. For extreme values, the sexes can also be distinguished based on tail length.

The São Tomé population of Lemon Dove presents a low sexual dimorphism in terms of coloration and morphometrics, with only the colour of the eye ring and, in most cases, the wing length differing between sexes. Sexual dimorphism in the São Tomé population of Lemon Dove has been previously described for plumage colour but not for eye ring colour (Naurois 1994, Leventis & Olmos 2009, Baptista et al. 2017). Our analyses of the photos of molecularly sexed individuals showed that plumage colour is an unreliable trait to sex the São Tomé populations of Lemon Dove. Plumage colour spans a wide continuum, both in males and females, varying from brownish to lighter grey and with different amounts of iridescence. Sexual dimorphism of plumage colour varies across distinct populations of Lemon Dove, with females generally duller (Baptista et al. 2017). As with the São Tomé population, the smallest individuals are female and the largest male, but there is a considerable overlap in size.

Contrarily to what is often assumed, the São Tomé Weaver does not have a clear sexual dimorphism based on plumage colour (Naurois 1994; Craig 2017). Morphometrics can only differentiate between the largest males and the smallest females. Plumage sexual dimorphism of their putative closest relative, the Brown-capped Weaver (Ploceus insignis), is restricted to the crown.

The São Tomé Thrush has a reduced sexual dimorphism. Males tend to have a longer wing, and females tend to be heavier. The only diagnostic character we could find for sexing adults was the colour of the iris: red in males, brown in females – a difference not mentioned in the literature (Collar 2017). The closest relative in the continent, the African Thrush (Turdus pelios), has no sexual dimorphism although it is unlikely that the eye colour has been analysed in detail as in here.

Non-sexual dimorphic species

The Principe Seedeater has almost no sexual dimorphism; the only morphometric characteristic that can pick up differences between sexes is the length of the wing at the extremes of the range. Most of the Principe seedeater African relatives are also non-sexual dimorphic (e.g., Crithagra leucopygia, C. canicapilla and C. gularis).

The São Tomé Prinia has no apparent sexual dimorphism contrary to what we found in the literature, but males do tend to have a longer tail, which allows sexing in the extremes of the range. The females have a similar, but somehow duller colour pattern in the head as it happens for most species in the genus (Ryan 2017).

As in the vast majority of the members of the 100+ species of the genus Zosterops, the São Tomé White-eye and the Black-capped Speirops have no sexual dimorphism (van Balen 2018). As in most cases studied here, males tend to be larger than females but the overlap is large.

Sex-discriminant functions analyses

The morphometric-based GLMs always allowed discriminating the sex of the Newton Sunbird, the Giant Sunbird and the Giant Weaver. They also allowed sexing the São Tomé Weaver (98.4%) with a high degree of certainty, and were only highly unreliable for the São Tomé White-eye (57.1%) and for the Black-capped Speirops (59%). The ability to distinguish between the sexes has increased considerably when considering altitude and habitat - which suggests that there is differentiation at very small scales, a subject requiring further studies. When the DFA can discriminate to 100 or almost 100% it means that there is a slight sexual dimorphism in allometry that we cannot detect by the eye or using single variables.

Calculating the accuracy of these functions to identify sex is overestimated when it is calculated using the same individuals that were used to build the model (Lachenbruck 1975). Therefore, the model should be validated using a different sample. This is normally calculated by the leave-one-out cross-validation, also known by jack-knife reclassification (White & Ruttenberg). However, having a small sample makes it difficult to do this. Nevertheless, the DFA is a great tool to sex birds, accomplishing better results than the methods based on simple unweighted combinations of variables, and should not be rejected so easily, despite the difficulties of implementation (Granadeiro 1993).

By adding both environmental variables, the São Tomé Weaver could also be sexed with full confidence. The inclusion of altitude alone allowed the correct sexing of São Tomé Prinia and São Tomé Thrush over 90% of the cases. This is strongly suggestive of micro-evolution taking place at very small scales on São Tomé for a wide range of species or, in alternative, could simply reflect a phenotypic response to different quality environments. The same species can occupy different habitats and doing so they can have slightly different changes in their morphometric measurements. This means that for these three species one needs to take into account the locality of capture (altitude and habitat) since this allows a considerable reduction in the overlap of measurements of males and

24 females. The general increase in size with altitude is consistent with Bergmann´s Rule (Salewski et al. 2009) and has been widely reported in birds. On the other side of the spectrum, the São Tomé White- eye and the Black-capped Speirops remain almost entirely indistinguishable. Even though, Moreau (1957) described the increase of size with altitude as a standard that applies to Zosteropidae both males and females increase in the same way and so remain sexually monomorphic.

Morphometrics

Morphometrics were diagnostic for sexing the Giant Weaver and the Giant Sunbird, and were able to sex the Newton Sunbird and the São Tomé Weaver in most cases. Morphometrics were completely non-informative for the São Tomé White-eye. Out of the four measurements used in this study, wing length was the most diagnostic measurement.

It has been shown that the overlap between male and female morphometric scores increases with sample size, until about 300 measurements (Mougin et al. 1986). Although the lack of data on molecular sexed individuals (since some species had a sample size less than 30 and none had more than 250 individuals molecularly tested), the current study revealed a wide overlap between the morphometrics of the two sexes in most species, making it difficult to use them as diagnostic characteristics of sexual dimorphism. If the sample increased it would not only confirm the overlapping but the tendency would be for it to increase. We could increase the number of molecularly tested individuals so the different morphometric values between sexes would be more accurate.

This study used a combination of data taken from several ringers, and thus might be affected by observer biases, especially for those measurements that are hardest to take, such as tarsus length. On the positive side, the differences we identified are sound enough to be detected even by less experienced ringers.

Conclusions

In São Tomé the birds are probably the best-known taxonomic group, but remarkably little research had been dedicated to improving our ability to distinguish the sex and age of the species for which sexual dimorphism is not immediately evident. This study gave us the required tools to a more assertive conservation management for the most common birds of São Tomé since it may allow us to understand how the different sexes use the territory and how they can be managed to ensure species survival. It allows a more accurate status of conservation of the species and it can also help to recognize characters to possibly be the subject of selection in the future.

Tropical birds are well known for their colourful appearance (Friedman & Remes 2016) but island environments share distinctive characteristics that underlie a worldwide pattern of parallel colour changes on island birds towards a reduction in brightness and colour intensity for both sexes (Doutrelant et al. 2016). One of the mechanisms that may be involved is the relaxation of selection on species recognition in these species-poor environments and/or a reduction in sexual selection (Doutrelant et al. 2016). This ‘loss of colour’ pattern has occurred in all our study species except for the São Tomé Paradise-flycatcher who seemed to be more colourful than its continent relatives.

Studies of sexual dimorphism can serve as an initial evaluation of the power of sexual selection and may help to recognize traits under selection (Berkunsky et al. 2009). If sexual selection is reduced on islands, it is expected to find less sexual dimorphism too (McLain et al. 1999; Doutrelant et al 2016). This might be illustrated by the Common Waxbill. Although not endemic, bill coloration differed from

25 the mainland populations in being slightly duller (more orange rather than red) and the plumage was more cream then white in the throat, albeit this was not tested.

The possibility of determining the sex and age of a species on the hand using measurements improves our ability to study population dynamics, behaviour, and better inform the management and conservation of populations. If the conservation status of species is only based on the number of individuals, there is a possibility of not detecting unbalanced sex ratios that may oppose recovery programs. In São Tomé there are a high number of people that hunt birds for human consumption (Carvalho et al. 2015). Knowing the sexes of the mostly hunted birds allows estimating the impact on the species’ demographics. If one of the sexes patrols its territory more often it can make it more vulnerable to hunters.

Determining whether different sexes use habitats and niches in the same way, may allow us to understand how the territory should be managed to ensure species survival. There is also the possibility of some birds living in different habitats in different stages of their life. Also, sex differences in juvenile mortality have been found in a variety of species, especially when food is scarce (Clutton-Brock 1986). This may be related with adult birds establishing their territory in optimal habitats and excluding the juveniles into sub-optimal habitats. This may for example be the factor driving the size differences with habitats that we detected here. Birds can also nest in specific areas that differ from their feeding areas. Although São Tomé has one protected area that covers 1/3 of the island it is not receiving the active conservation management required and understanding if some species depend on different parts of the park for different stages of their life-cycle may be important for their survival (Soares 2017).

Knowing the sex and age of the birds can show us where the weaknesses of the population lie, and direct the conservation effort where it should be applied. This work gave us the required tools to confidently sex seven endemic species and a non-endemic of São Tomé, allowing the developing of better informed conservation strategies.

CHAPTER 2: Seasonality in the tropics: Breeding and moulting of the common birds of São Tomé Island

Abstract: Organisms have evolved to time their reproductive efforts to match the peak of resources required to raise their offspring. In temperate areas this has led to narrow breeding seasons coincident with a marked seasonality in resource availability. In contrast, tropical areas close to the Equator are some of the most stable environments of Earth, with little seasonality in resource availability. This has suggested that rainforest species are able to breed throughout most of the year. On another hand, variation in precipitation could lead to changes in resource availability affecting breeding decisions. The scarcity of detailed studies in breeding activity in rainforest species has prevented to effectively test this, even in birds – one of the best studied vertebrate groups. Here we conduct a detailed study on the breeding and moulting seasonality of São Tomé common bird species, linking them to environmental constraints and species traits. The breeding season and time of moult was identified for each species, based on ringing data and focusing on the 12 species with the highest number of records. The breeding activity of São Tomé common bird species occurred mostly during the rainy season, peaking during the short dry season of gravanito. The moulting followed breeding activity, and still occurred mostly before the start of the long dry season, the gravana. Heavier species tended to breed earlier, probably due to having longer breeding seasons and to ensure that hatching coincides with peak food availability. Our results also suggest that moulting, and thus breeding, are delayed at higher altitudes. This information is key to support bird conservation strategies, namely regarding hunting and other extractive activities. It also shows that, despite reduced environmental variability, the annual life cycle of tropical birds might be structured around precipitation-driven changes in resource availability.

Keywords: endemism, environment, GLM, rainfall, reproduction strategies

INTRODUCTION

Almost all organisms experience some seasonal variations in their environment such as variations in rainfall, temperature, food abundance and disease (Nelson & Demas 2004). Seasonal changes increase with increasing latitude, becoming almost imperceptible in the equator. Birds are the vertebrate group for which more ecological data, including on reproduction, is available, allowing studying how breeding decisions change with latitude/seasonality (Komdeur 1996; Salewski et al. 2010). Still, most studies on bird reproduction come from temperate areas, with very little data available from the rainforest communities, the most stable environment on Earth. Nevertheless, the effort to comprehend the variation of seasonality within tropical populations and among species in tropical assemblages has been quite small (Stouffer et al. 2013).

Reproduction is a fundamental feature of all known life. Reproduction is very demanding in terms of energy; therefore organisms need very accurate timing to breed when environmental conditions are favorable (Martin & McKay 2004). Moreover, breeding at the correct time can also decrease weather- related nest failures, such as flooding burrows and saturation of open cups by heavy rains, risks that can explain the importance on timing of breeding (Skagen & Adams 2012). Bird populations present very different breeding seasonality depending on the region, habitat, and more specifically on the climate variations of the region they occupy (Martin & McKay 2004; Hau et al. 2008). Generally, breeding timings tend to be shorter in temperate zones and longer in the tropics (Moore et al. 2005). Most species in temperate areas fine-tuned their breeding periods to coincide with the short periods

27 favorable for reproduction by making use of the precise variations in photoperiod to start the breeding season (Wikelski et al. 2000). In the tropical regions close to the equator (‘rainforest biome’) food supply is regular throughout the year and overall environmental conditions are ideal for survival. Hence, rainforest species tend to have more relaxed breeding seasonality, taking advantage of the wider periods with favorable environmental conditions (Nelson & Demas 2004). Changes in the photoperiod of tropical areas are often considered too insignificant and it is presumed that rainfall is used as the short-term seasonal cue (Komdeur 1996).

The breeding of birds can be detected by changes in behavior, plumage and physiology, but the presence of a brood patch is one of the easiest and most reliable means to confirm breeding activity (Tucker 1943). The brood patch is a featherless, reddish, and wrinkled skin with many blood vessels visible on the underparts of most breeding bird species, which develops in the ventral region before incubation to improve heat transfer to the eggs (Demongi 2016). As a rule, it develops only in females, but in species where males incubate, these can also develop a semi-patch and more rarely a full brood patch. A few days after hatching, a gradual return to the normal condition will start, marked by a decrease of the wrinkles and a return of the transparent skin (Svensson 1992).

The timing and stage of plumage moult is another important cue on the breeding period. Moult is the regular replacement of all or parts of the plumage required to replace damaged or lost feathers, thus maintaining the functions of plumage. Breeding and moulting are the primary periods of the annual cycle in most birds. Both actions are costly in terms of energy; therefore usually they do not overlap in time (Demongin 2016; Freed & Cann 2012). The initiation of moult is correlated with the breeding season, so much so that the end of the breeding season is assumed to begin with moult for most species of birds (Jenni & Winkler 1994). Moulting strategies can vary between species and the actual timing of moult may vary for different populations of the same species (Svensson 1992). In some species it is also used to adjust plumage characteristics to specific life cycle needs of the bird like the first complete moult of juvenile birds that acquire the adult plumage and post-breeding moults in the end of the reproduction cycle (Jenni & Winkler, 1994).

The island of São Tomé is an important center of bird diversity and endemism (Jones & Tye 2006). Compared to mainland counterparts, island birds tend to have lower fecundity, greater reproductive investment and extended developmental periods (Covas 2011). Less is known about the extent and variability of the breeding season, parameters which are vital for understanding population dynamics, and to assess the causes behind demographic changes (Burr et al. 2016).

This chapter focuses on determining the breeding and moulting seasonality of São Tomé common bird species, using a large long-term dataset of birds captured in the island. More specifically, we will describe breeding and moulting seasonality and link them to species traits, seasonal rainfall variability, altitude and habitat type. By contributing towards a better understanding of seasonality in São Tomé, we will be providing key information to guide much-needed evidence-based conservation actions for the island, and for similar tropical island ecosystems since not much is known on how tropical birds cope with seasonal variations in their environment.

METHODS

Study Area

São Tomé (00°25’N–00°01’S, 06°28’E–06°45 E) is the largest island (857 km²) of the Democratic Republic of São Tomé and Príncipe. Located the Gulf of Guinea (West Africa – Fig. 2.1) it is part of the Cameroon Volcanic Line. It is about 45 km long and 32 km wide, having a maximum altitude of 2024 m at Pico de São Tomé. São Tomé is c. 150 km apart from Príncipe Island and 250 km from the African continent. This location allowed high levels of bird endemism, as it is far enough from other land masses to be isolated, but near enough to be colonized by many distinct taxa (Melo & O’Ryan 2007).Uninhabited and covered by native rainforest until 500 years ago, this tropical island offers an excellent opportunity to investigate the ability of species to adapt to life on an island with and without anthropogenic environmental changes (Atkinson et al. 1991).

The prevailing southwest winds and the steep mountains create a marked rainfall gradient, annual precipitation reaching over 7000 mm in the southwest, but less than 600 mm in the northeast (Jones & Tye 2006). There are two main seasons in São Tomé: the dry season (gravana), between June and August, and the rainy season, from September to May. There is also a smaller dry season called gravanito, between December and January (Jones & Tye 2006). The annual average temperatures range between 18°C and 33°C, decreasing with rainfall, altitude and during the dry season, even though the average temperature is fairly constant throughout the year. Humidity levels and cloud cover are also fairly high and constant throughout the year in most of the island (Carvalho et al. 2004; Jones & Tye 2006).

São Tomé native forest is rich in endemic species and has a dense canopy, with trees exceeding 30 m of height, with an abundance of lianas and epiphytes, such as mosses, ferns and orchids (Peet & Atkinson 1994). Most of the lowland forest was cleared for sugar cane by the mid-16th century, with a second bout of deforestation taking place in late 19th century, when significant areas were cleared up to 1200 m to plant export crops, such as coffee and cocoa. Subsequent agricultural abandonment led to significant regeneration (Tenreiro, 1961; Oliveira 2002, Carvalho et al. 2004). The resulting secondary forests, some of which also resulted from extensive logging, have lower canopy, higher proportion of introduced species and less biomass than native forests (Exell 1944).

The ecosystems of São Tomé are marked by remarkable climatic and altitudinal gradients. The dry areas of the northeast are subject to frequent fires, have many deciduous tree species (Leventis & Olmos 2009), and have been extensively cleared for agriculture, so much so that much of it has been turned into an anthropogenic savannah (Oliveira 2002; Carvalho et al. 2004). On other hand, the rainforest, which still covers most of the central and southern half of the island, is stratified in: lowland forest, ranging from sea level to 800 m; montane forest, from 800 to 1,400 m, and mist forest from 1,400 to 2,024 m (Exell 1944).

São Tomé remains little studied, despite being home to a wide variety of endemic and endangered species from several taxonomic groups, such as land mollusks, orchids and birds (Jones 1994). Twenty out of the 50 terrestrial bird species that breed on the island are endemic to the Gulf of Guinea, nine of which are globally threatened, and heavily reliant on native forest.

Most of the native forest is included in the Obô Natural Park, which covers 244 km2 of the island (Dallimer et al. 2009).

Data collection

We compiled data from bird ringing taking place in São Tomé Island since 2002 by Martim Melo and colleagues. These were collected throughout the year in 30 sampling locations across the island: Abade; Alto Douro; Angus; Bom Sucesso; Calvário Bend; Chamiço; Contador dam; Duas Grotas; Esperança; Governador; Jalé; Lagoa Amélia; Lembá camp; Macambrará; Margão; Martim Mendes; Monte Café; Monte Carmo; Monteforte; Morro Artur; Morro Claudina; Mucumblí; Nova Moca; Pico Calvário; Praia Cruz; Quija; Roça São João; S. Miguel; Umbumgu; Vanguarda (Fig. 2.1).

We collected additional data between mid-January and the end of March of 2017, coinciding with the gravanito and the restart of the rainy season. The birds were captured using mist-nets set at understory level (0.5–4.0 m above ground). The number and location of the nets were decided in situ to maximize the diversity and number of birds being captured, while assuring we had the capacity and time to process them in safety (De Beer et al. 2001). Whenever possible, nets were opened at 05.00 am, just prior to dawn, and kept open until 05.30 pm, although timing was frequently constrained by logistical factors, like weather and accessibility. Each location was sampled for three days in a row, the ideal period to minimize mist-net avoidance (Marques et al. 2013, tested locally). All birds captured in the mist-nets were identified to species level and ringed. Standard measurements were taken and it was assessed if birds had an active brood patch and/or were moulting primary or secondary flight feathers (De Beer et al. 2001). A brood patch was considered active when the ventral surface was completely or almost completely naked, and the skin was thickened, wrinkled and reddish due to the abundant superficial blood vessels (Svensson 1992). We added our records to the São Tomé bird ringing database, which was then scanned for mistakes, and standardized.

Data analysis

To assess seasonal patterns, we identified the breeding season and time of moult for São Tomé common bird species. This was done by counting the number of species that had a brood patch or were moulting in each month, respectively. These patterns were compared to monthly rainfall (Instituto Nacional de Meteorologia de STP) in order to understand the link with seasonal weather changes. Subsequently we assessed the breeding and moulting seasonality of each species, by counting the number of individuals of each species that had a brood patch or were moulting in each month, respectively. Since males usually do not form brood patches (Demongin 2016, confirmed in situ), we tried to explore breeding seasonality just looking at the proportion of females with brood patch, but due to difficulties in sexing individuals and lack of data (Fig. S5)(Madeira 2018) we decided to take into account the overall proportion of individuals with brood patch.

The peaks of the breeding and moulting seasons were determined as the median date in which individuals with brood patch and moult were detected, respectively. We used Spearman's rho to assess if the timing of breeding and moulting season peaks were related to mean mass and forest dependency of common bird species. The mean mass was calculated as the average of the measurements collected during fieldwork, while forest dependency was calculated as the proportion of records in point counts in native forest, corrected for sampling effort (Soares 2017).

Since there was not enough brood patch data, we used Generalized linear models (GLM) to assess the influence of species, habitat and altitude on moulting seasonality. This analysis focused on the eight species that had at least 200 records with information on habitat and altitude (Table 2.1).

Sampling locations were categorized as forest or plantation, and as montane or lowland, based on whether they were located above or below 800 m a.s.l., respectively. To identify significant variables and interactions, we used chi-square tests and a stepwise backward elimination procedure on the full model (Griffin et al. 2014). To identify important variables in explaining the proportion of moulting individuals in each month, we calculated the relative variable importance, which applies model averaging to the “dredge” function of the pre-selected model (Barton 2018). Both model selection procedures use Akaike Information Criterion corrected for small sample size (AICc).

RESULTS

Our study focused on the 12 species that had more than 100 records, to ensure we could perform robust statistical analyses (Table 2.1). Since 2002, we obtained 5669 records of these species across São Tomé Island (Fig. 2.1), of which 738 were collected in 2017. Henceforth, these species will be referred to as ‘São Tomé common bird species’. Some abundant species were left out because their capture rate was low. From the 5669 records, 223 had an active brood patch and 916 were moulting. To better visualize the data of the total brood patches and moults we compiled this information in graphics (Fig. S3 & S4). 3830 individuals were captured on forest and 1839 on plantation; 3316 on montane and 2353 on lowland regions.

Table 2.1- List of the common birds of São Tomé. Bird characteristics, including the number of valid records and key species traits such as mean mass (from our database), conservation status (IUCN 2017), forest dependency and trophic guild (Soares 2017).

Forest Mass Conservation Trophic English name Scientific name Endemism dependency n (g) status guild (%)

Newton's Anabathmis newtonii 6.9 17.1 662 Sunbird Endemic LC Omnivore Columba larvata 173.2 19.1 139 Lemon Dove (simplex) Endemic LC Omnivore Crithagra 23.4 16.8 462 Principe Seedeater rufobrunnea Endemic LC Omnivore Giant Sunbird Dreptes thomensis 24.4 Endemic VU Omnivore 73.8 136 Common Waxbill Estrilda astrild 7.7 Non-endemic LC Granivore 0.1 254 Giant Weaver Ploceus grandis 60.6 Endemic LC Omnivore 2.3 126 S.T. Weaver Ploceus sanctithomae 21.1 Endemic LC Omnivore 18.0 347 S.T. Prinia Prinia molleri 9.6 Endemic LC Insectivore 17.1 340 S.T. Paradise- Terpsiphone 13.5 17.7 485 flycatcher atrochalybeia Endemic LC Insectivore S.T. Thrush Turdus olivaceofuscus 84.1 Endemic NT Omnivore 23.7 882 S.T. White-eye Zosterops feae 8.1 Endemic VU Omnivore 10.5 109 Black-capped Zosterops lugubris 16.9 17.9 1727 Speirops Endemic LC Omnivore

Figure 2.1- Map of São Tomé Island with sample location. The pie charts shows which species were captured in each sampling location. Each species listed in the legend is always in the same portion of the pie chart, in counterclockwise order starting from the top of the pie chart. The portions of the pie chart are coloured if the corresponding species occurred at least once at that location. The size of the pie chart is proportional to the number of records, in a logarithmic scale. The initial 37 sampling locations were grouped in 23 map locations to allow visualization, since some of them were in close proximity of each other.

Breeding and moulting activity

Reproduction and moulting of São Tomé common bird species was detected in all months of the year, with the exception of July and August for moulting, but seasonality was still detected and coincided with the rainy season (Fig. 2.2). The number of common bird species with active brood patches increased between October and May, peaking in January, when 11 out of 12 species were confirmed breeding. The number of common bird species moulting increased more gradually, starting with only two species in September, and peaking between March and May, when all species were moulting.

Figure 2.2 – Link between rainfall, breeding and moulting seasonality in São Tomé. Monthly rainfall throughout the year is represented by the blue line (INM), the number of species breeding by the red columns and the number of species moulting by the green columns.

When we compare each species individually we can see that, despite some species-specific variation, this pattern is maintained on almost all of our study species: they showed little breeding activity during the gravana (May to September) and had their peak of moulting after the breeding season, which culminates during the gravanito (January to February) (Fig. 2.3). Our female-based analyses did not cover all months which may have biased the results (Fig. S5).

Figure 2.3 – Breeding and moulting activity of São Tomé common bird species. From the species that breeds the earliest to the one that breeds the latest (with the exception of the Lemon Dove Columba larvata). The graphs start in June. The percentage of breeding individuals in each month is represented in red, while that of moulting individuals is shown in green. The thickness of the columns represents the proportion of individuals sampled, compared to the month with most individuals sampled for each species. The grey columns highlight months without sampling of the respective species.

Species traits and seasonality

Heavier species tended to breed earlier (rho = -0.73, p-value = 0.015, R2 = 0. 33 – Fig. 2.4), but there was no clear correlation between moulting time and mass (rho = -0.11, p-value = 0.733, R2 = 0.01). There was also no clear correlation between native forest dependency and breeding (rho = 0.31, p-value = 0.356, R2 = 0.05) or moulting periods (rho = -0.08, p-value = 0.800, R2 = 0.05).

Figure 2.4 – Correlation between the timing of breeding (red) and moulting (green), and species traits: a) mean mass and b) forest dependency, between the end of October (Oct) and the end of March (Mar).

Environmental characteristics and seasonality

GLMs revealed that the interaction between altitude and month was important to explain the proportion of moulting individuals, and that birds tend to moult later in montane areas (Fig. 2.5). We could not fit a binomial GLM to our data, but the use of linear models showed that this was the only significant interaction, and that the use of a quadratic term on the month did not improve the models (Table 2.2). The modelling also showed that the proportion of moulting individuals varied between species, altitude and habitat.

Figure 2.5- The monthly proportion of moults (mt) in altitude for the eight species with the most records. The red circles represent the proportion of moulting individuals in montane areas, and the black circles the proportion in lowlands. The months are represented from July to June. 35

Table 2.2 – Comparison of models built to explain the monthly proportion of moulting individuals for each species. The Chi-square test results from stepwise backward elimination are shown, together with the AIC of the most complete model for each comparison. The last model represents de AICc.

Model comparison AIC P-value

glm(mt~I(month^2)+sp*month*habitat*alt,data=month2)<- 125 <- 0.68 glm(mt~sp*month*habitat*alt,data=month2) 123

glm(mt~sp*month*habitat*alt,data=month2)<- 0.60 glm(mt~sp+month*habitat*alt,data=month2) 89.5 glm(mt~sp+month*habitat*alt,data=month2)<- glm(mt~sp+month*habitat+month*alt,data=month2) 85.8 0.84

glm(mt~sp+month*habitat+month*alt,data=month2)<- 85.5 0.21 glm(mt~sp+month+habitat+month*alt,data=month2)

glm(mt~sp+month+habitat+month*alt,data=month2)<- 88.5 0.03 glm(mt~sp+month+habitat+month+alt,data=month2) glm(formula = mt ~ sp + month + habitat + month * alt, data AICc = month2) 87.6

The model averaging revealed that month (relative variable importance – RVI = 0.92) and altitude (RVI = 0.79) were the most important variables to explain the proportion of moulting individuals, followed by habitat (RVI = 0.68), the interaction between month and altitude (RVI = 0.61) and species (RVI = 0.52 - Fig. 2.6).

Figure 2.6 – The cumulative Akaike weight of all possible linear models used to explain the proportion of moulting individuals, based on the best model from Table 2.2, which includes altitude (alt), habitat, month, species (sp) and the interaction between altitude and month. The models are ranked by increasing AICc. Each row represents a model and each column represents a variable. A full cell indicates that a certain variable is present in a model, and the taller the cell the better the model, based on weight, which is in itself calculated based on AICc changes between models. 36

DISCUSSION

The breeding activity of São Tomé common bird species was detected in all months of the year which is in agreement with the environmental stability found on tropical areas close to the Equator with show little seasonality in terms of resource availability. However, it was possible to detect a peak of reproduction matching the mid-point of the rainy season marked by the short dry season, the gravanito. This suggests that rain, do lead to an increase in food resources required for the feeding of the young, and in particular of the insects that chicks need to grow rapidly. Moulting followed breeding activity and occurred mostly before the start of the long dry season, the gravana. Heavier species tended to breed earlier, and moulting seems to occur first in the lowlands.

Breeding seasonality

Most common bird species showed breeding activity from October to May, peaking around the gravanito in January/February. Five of our study species have a long breeding season, which lasts longer than seven months. Five species had breeding seasons that lasted at least five months. We could not define the breeding season for the remaining two species, mostly due to the small sample size.

Overall, these results are consistent with previous knowledge, although a few differences are worth mentioning. The São Tomé Speirops and the São Tomé Thrush showed a long period with brood patch, even though other authors claim that the breeding season of the Speirops has a later start and a shorter breeding seasonality. As for the Thrush it starts a month earlier than showed on our data (Naurois 1994; Jones & Tye 2006; Leventis & Olmos 2009; van Balen 2017). The Principe Seedeater, the Giant Sunbird and the Giant Weaver also have an extended breeding season, despite previous works mentioning smaller breeding seasons for the last two species (Naurois 1994; Atkinson et al., 1991; Leventis & Olmos 2009; HBW Alive 2017). None of these breeding periods seems to be interrupted, so we argue that, unlike what has been claimed before for these species, there is a single breeding season. However, this is long enough to allow multiple clutches.

The Newton Sunbird, the São Tomé Weaver, the São Tomé Prinia, the São Tomé Paradise- flycatcher and the São Tomé White-eye have breeding seasons that last five or six months. The Newton Sunbird had his peak of breeding in January, as already suggested by Atkinson et al. (1991). It had been suggested that the breeding of this species occurs throughout the year (Robert et al. 2001) but our data did not support this. The São Tomé Weaver starts breeding a month earlier than the Newton Sunbird as previously reported (Leventis & Olmos 2009, Craig 2017). The São Tomé Prinia breeds between October and February, having its peak in January, even though the literature suggests a longer breeding season (Naurois 1994; Jones & Tye 2006; Leventis & Olmos 2009; Ryan 2017). We detected brood patches in the São Tomé Paradise-flycatcher between October and February, suggesting a breeding season only slightly shorter than previous records (Naurois 1994; Leventis & Olmos 2009; Moeliker 2018). The São Tomé White-eye breeds between November and March, peaking in February. The smaller number of records for this species makes it harder to assess seasonality patterns, and it might start earlier (Naurois 1994), even though the breeding biology of this species is particularly little understood.

The breeding seasons of the Lemon Dove, Common Waxbill and Giant Sunbird could not be clearly defined for different reasons. The Lemon Dove does not develop a brood patch, but the few fledgings we caught and the moulting seasonality suggest that the breeding season occurs between September and February, which coincides with what had already been described (Naurois 1994; Jones & Tye 2006; Leventis & Olmos 2009; Baptista et al. 2017). The Common Waxbill reproduction seems to peak in mid-January, but only ten out of the 100 brood patch checks for this species were taken 37 during the seven months between April and October, making it difficult to assess if it nests throughout the year, as it has been suggested (Naurois 1994; Leventis & Olmos 2009). The Giant Sunbirds have few abdominal feathers and a thick skin, which we believe might have led to false positive records of brood patch outside the breeding season.

The unbalanced sex ratio of captured birds and the uneven sampling effort throughout the year have limited the interpretation of breeding seasonality from brood patches. The males made up over 60% of the captures for the Newton Sunbird, the Principe Seedeater, the São Tomé Weaver and the São Tomé Thrush, obscuring our ability to calculate the proportion of breeding individuals, especially in species that are hard to sex (Madeira 2018). Since the analyses of brood patch seasonality are only carried out on females, putting together a database with more molecularly sexed female individuals would greatly improve our knowledge on the reproductive seasonality of São Tomé bird species – especially on those that are difficult to mist-net or for which brood patches are not easily identifiable, such as the Lemon Dove or the Giant Sunbird. Since compiling observations on other aspects of reproduction, such as nest-building, egg-laying, and hormonal changes, are very difficult to collect in the São Tomé Island, the presence of brood patches and description of moult stage, and any records of juveniles or fledgings, will continue to be the best cues to determine breeding seasonality. We think that the limitations reported here relate more to sampling size than to the methods per se. In this regard, April was the least sampled month, causing an obvious scarcity of brood patch records when compared to the neighboring months.

Overall, the breeding season of common bird species in São Tomé was influenced by precipitation, as occurs for their continental relatives (Alive HBW 2017). For example, the mainland relatives of the sunbirds species have a breeding season that goes from January to June and from October to December, which coincides with the two peaks of rainfall in Cameroon (Cheke & Mann 2017).

Moulting seasonality

Most common bird species had their peak of moulting during the gravanito, or soon after it. The Principe Seedeater, the São Tomé White-eye and the São Tomé Speirops had their peak of moulting the month after they had their breeding peak. The São Tomé Paradise-flycatcher and the São Tomé Thrush had it two months after; the Newton Sunbird and the São Tomé Prinia had it three months after; the Giant Sunbird, the São Tomé Weaver and the Giant Weaver had it four months after. The breeding and moulting peaks coincided in the same month for the São Tomé White-eye and Common Waxbill, both of which were less sampled than other species. Our data are in agreement with the information collected by King & Dallimer (2003) and Jones & Tye (2006). According to the latter, the São Tomé Prinia was moulting on August, a month for which we have no data for this species.

Little is known about the moulting strategies of São Tomé birds, but our results strongly indicate most species have a single adult post-breeding moult of their primaries. Some species might have a partial pre-breeding moult to acquire breeding plumage like probably the male of the Giant Weaver. Recording moults in greater detail would be key to fully understand moulting strategies, and to assess species-specific differences.

Some study species appear to be moulting and nesting at the same time in the same location, which does not mean that the same individual is going through these processes simultaneously. Breeding and moulting accrue large energy costs, and it is thus more likely that each individual only starts moulting after breeding, and that there are slight mismatches between the timings of different individuals (Tarroux et al. 2003; Jones & Tye 2006). The long moulting periods we registered for most species might be linked to seasonal differences across the island or between years, which would compromise

38 our ability to interpret the results in detail, namely regarding the overlap between breeding and moulting periods.

Phenological determinants: environmental characteristics and species traits

Birds seem to time breeding and moulting so as to avoid the gravana, the main dry season, which suggests that this is a period of resource scarcity in São Tomé (Jones & Tye 2006). The bird breeding and moulting seasonality we described are in line with the peak flowering, fruiting and insect activity seasons, all of which occur during the rainy season, and especially around the gravanito (Bancroft et al. 1999). Since the food availability is directly correlated with increased rainfall we can say it is the main reason to affect bird phenology (Hau et al. 2008).

The larger species tended to breed first, which makes sense since they also take longer to reproduce, and would thus need to start breeding first to synchronize the hatching with the most favorable time of the year to raise the offspring (Verhulst & Nilsson 2008). We did not detect phenological differences based on forest dependency, but more detailed studies might detect subtle differences between habitats. The breeding season should also be affected by food preferences (Hau et al. 2008), but most sampled species were omnivore, making it difficult to detect differences.

We used modelling to understand the effect of species, habitat and altitude on breeding seasonality. Since brood patch data was scarce, we focused on moulting data, which we know happens after breeding, and verified that moulting tends to be delayed in montane areas compared to lowlands. Altitude is associated with lower temperatures leading to seasonal delays in the peaks of insect and fruit abundance (Bears et al. 2009). Correspondingly, bird breeding was also delayed at higher altitudes, this may explain the long breeding seasons of some species being a joining of several different breeding seasons. The model we built tried to identify the most important variables to explain the proportion of moulting individuals for each month, and since we only had robust data for eight species, the best model we could create was not without faults. Namely, it failed to include a quadratic term for month, when it was obvious that the link between moulting was non-linear, or a generalization using the binomial family, which would certainly be more appropriate, since the response variable was a proportion. It is thus remarkable that the altitudinal delay in breeding could still be detected.

Implications for conservation

This study made progress in understanding how tropical birds cope with seasonal variations in their environment by specifically providing a better understanding of the breeding seasonality of São Tomé common bird species. Besides being key to understand the ecology and behavior of these species, this knowledge is key to develop conservation strategies and to implement conservation initiatives. The distribution of birds in São Tomé has been shown to be negatively influenced by hunting (de Lima et al. 2013; Carvalho et al. 2014; de Lima et al. 2017), activity whose impact is higher when coincident with breeding activity. This coincidence has been reported for the Island Bronze-naped Pigeon Columba malherbii (Carvalho 2015). Knowing the critical breeding time for the species of São Tomé is key to develop awareness and population management strategies that ensure that the endemic-rich avifauna of the island can persist and thrive.

Improving our knowledge on the breeding seasonality of São Tomé birds allows us to assess how ongoing climate change might change breeding seasonality, and affect breeding success, thus constituting a tool to understand how climate change can affect the more sensitive species in tropical habitats. Forests on tropical islands can be affected in different ways by climate change. The

39 increasing storm frequency is of particular concern in São Tomé since variability in rainfall can result in disturbances in the ecosystem. This rain irregularity can alter the breeding season of birds in São Tomé since it is one of the cues to start reproduction, and it can also change food availability that will jeopardize breeding success in the island (Hau et al. 2008).

The rare endemic species are thus likely to be facing growing pressures in the nearby future, as the quality of the remaining forest will continue to be negatively affected by introduced organisms and direct anthropogenic ecosystem degradation (Dallimer et al. 2009; Vásquez et al. 2017).

Improving our knowledge on the breeding seasonality of São Tomé birds also allows us to better understand the insular patterns and compare them between mainland birds based on both endemic and nonendemic species.

FINAL CONSIDERATIONS

Islands are recognized as highly important for biodiversity and as such are often recognized as priority conservation areas (Paulay 2015). This study complements the knowledge on breeding strategies of island assemblages. Namely it focuses on the sexual dimorphism and breeding seasonality of São Tomé common bird species, which is crucial both for fundamental research on ecology and evolution and for guiding conservation strategies of endemic-rich island biodiversity.

In the first chapter we tried to better determine the sex and age of São Tomé common species on the hand using measurements and coloration patterns. This work gave us the required tools to confidently identify the sex of seven endemic species and a non-endemic of São Tomé and coloration helped in the identification of the age in most species. This can improve our ability to study population dynamics and behavior allowing the development of better informed conservation strategies, since it may allow us to understand how the different sexes use the territory and how it can be managed to ensure species survival. It allows a more accurate status of conservation of the species and it can also help to recognize characters that will possibly be the subject of selection in the future.

In the second chapter we tried to understand if there was a breeding seasonality on São Tomé common bird species. We found that the breeding activity of São Tomé common bird species occurred mostly during the rainy season, peaking during the short dry season of gravanito. Moulting followed breeding activity and heavier species tended to breed earlier, as expected from their longer breeding seasons. Our results also suggest that moulting, and thus breeding, are delayed at higher altitudes, associated with lower temperatures leading to delays in the peaks of insect and fruit abundance (Bears et al. 2009). This study made progress in understanding how tropical birds cope with seasonal variations in their environment and this is key to develop population management strategies that ensure that the endemic-rich avifauna of the São Tomé Island can persist and thrive.

The evidence obtained in this study is key to support bird conservation strategies, since not much is known on how tropical birds cope with seasonal variations. The distribution of birds in São Tomé has been shown to be negatively influenced by hunting (de Lima et al. 2013; Carvalho et al. 2016; de Lima et al. 2017), activity whose impact is higher when coincident with breeding activity. This study also constitutes a tool to understand how climate change can affect the more sensitive species in tropical habitats since it has shown that precipitation patterns are the main cue to start reproduction, and rain levels can also change food availability that will jeopardize breeding success in the island (Hau et al. 2008). Understanding these impacts, coupled with determining the mosaic of habitats needed to support endangered wildlife in tropical areas that are subject to rapid economic change, is essential for the conservation management (King & Dallimer 2003). The Obô national park is likely to be facing growing pressures in the nearby future, as the quality of the remaining forest will continue to be negatively affected by the rapid economic change and fast population growth. Applying conservation actions in the afected forests is crucial because they are the stronghold of the unique biological communities of the island and so they are of relatively great importance for conservation of the São Tomé avifauna. We expect to see a resurgence of international interest in the biodiversity of São Tomé Island, which could stimulate renewed efforts to ensure the long-term conservation of these unique endemic species.

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SUPPLEMENTARY MATERIALS

Figure S1- Bi-plots of five morphological measurements (Mass-Wing; Mass-Tail; Mass-Tarsus; Wing-Tail; Tail- Tarsus) for 11 endemic bird species of São Tomé and for one non-endemic. The species are ordered alphabetically, according to the scientific name.

Figure S2- São Tomé Thrush differences in iris colour between males and females of molecularly tested individuals. The males are represented by the black circles and the females by the red ones.

Figure S3 - Brood patch sample effort distribution, throughout the year. The graphs start in June. The total number of captured individuals in each month is represented in blue; the red column represents the number of individuals assessed for brood patch and the green one represents the number of birds identified with brood patch. The Gray column represents unsampled months.

Figure S4 - Moult sample effort distribution, throughout the year. The graphs start in June. The total number of captured individuals in each month is represented in blue; the red column represents the number of individuals assessed for moult and the green one represents the number of birds identified with moults. The Gray column represents unsampled months.

Figure S5 – Breeding and moulting activity of the sexed females of the 12 common bird species of São Tomé. From the species that breeds the earliest to the one that breeds the latest (with the exception of the Lemon Dove Columba larvata). The graphs start in June. The percentage of breeding individuals in each month is represented in red, while that of moulting individuals is shown in green. The thickness of the columns represents the proportion of individuals sampled, compared to the month with most individuals sampled for each species. The grey columns highlight months without sampling of the respective species.