Testing Phenotypic Plasticity in a Potential Ecological Speciation

TESTING PHENOTYPIC PLASTICITY IN A POTENTIAL ECOLOGICAL SPECIATION

SCENARIO: THE GORGONIAN CORAL Antillogorgia bipinnata (Cnidaria: Octocorallia)

Iván F. Calixto‐Botía & Juan A. Sánchez*

Departamento de Ciencias Biológicas-Facultad de Ciencias, Laboratorio de Biología

Molecular Marina (BIOMMAR), Universidad de los Andes, Carrera 1E No. 18A-10, Bogotá,

Colombia. *Corresponding author: [email protected]

ABSTRACT

Phenotypic plasticity, as a non-heritable response induced by the environment, has been proposed as a key factor in the evolutionary history of corals. In the Caribbean, a significant number of octocoral species show strong phenotypic variation, with most species exhibiting a great overlap in intra- and inter-specific morphologic variation preventing us to understand their evolutionary history. This is the case of the gorgonian octocoral

Antillogorgia bipinnata (Verrill 1864), which shows three polyphyletic morphotypes along a bathymetric gradient. The morphotypes are discernable along a continuum with characteristic traits in colony height, length, branch arrangement, internodal distances, and sclerite shapes (calcite skeletal structures). This research sought to test phenotypic plasticity in A. bipinnata by a reciprocal transplant experiment involving 256 explants (32 colonies) from two morphotypes in two locations (15 km apart each other) and two depths

(2-3m and 7-8,5m) in Bocas del Toro, Panama. Horizontal and vertical growth, number of new branches and internodal distance were compared after 13 weeks of transplant. The data were processed under a Generalized Linear Mixed Model showing correlation between the target habitat and each of the traits evaluated. The overall growth rates behaved similar to native transplants. Internodal distances for foreign transplants were higher for ‘deep habitat’, and shorter for ‘shallow habitat’. Particularly, nonparallel reaction norms between numbers of new branches indicate variance due to a genotype-environment interaction.

Additionally, the spawning patterns of native morphotypes were examined, indicating reproductive asynchrony between habitats. These results support a scenario where adaptive plasticity, a genetic component and a plausible mechanism for gene flow reduction between morphotypes would shape a potential process of ecological speciation related to a depth cline in A. bipinnata.

Keywords: phenotypic plasticity, reaction norm, octocoral, depth cline, spawning synchrony.

INTRODUCTION

Phenotypic plasticity is the natural capacity of a genotype to react with a phenotypic change due an environmental variation. It has been understood as a barrier to speciation due to the intuitive idea that if there is no need for genetic change to adapt to the environment (masking the genotype for negative selection), then the process of adaptive genetic divergence will be hindered [1,2]. However, the potential role of adaptive plasticity has been suggested in some cases where it can promote speciation, like contributing to niche diversification and further evolutionary change [3,4,5,6]. Phenotypic and genetic accommodation, the Baldwin effect and the Waddington’s genetic assimilation are mechanisms that within the classical Mendelian inherence have the power to explain environmental-induced changes fixed in the genome and susceptible to promote an speciation process [7,8].

Reciprocal transplant, or common garden, experiments consist of moving the phenotypic variants to the opposite environments and are still a practical and cost-effective approach to test phenotypic plasticity. In these experiments the direction and degree of response to environmental factors are assessed in a graphical representation of reaction norms of the traits across different environments [9], thus identifying genotype and environment interactions [10]. In marine systems, variation related to environmental heterogeneity has been particularly studied along the depth gradient using reciprocal transplant experiments.

This continuum provides marked physical and biological variation in parameters such as light intensity, water motion, predation and suspended particles [11].

In corals most studies assessing plasticity and genetic adaptation have shown adaptive divergence linked to the depth gradient. One of the first extrapolations from the common garden methodology on corals was done with Orbicella annularis and Siderastrea siderea by

Foster (1979), in four reef habitats, detecting high phenotypic plasticity responding to the transplanted habitat [12]. West et al. (1993) and West (1997) assessed two morphotypes responding to a depth cline in the octocoral Briareum asbestinum employing natural and artificial reciprocal transplants (i.e. in lab simulation of environmental parameters). Traits assessed showed an adaptive response of genotype x environment interaction, highlighting the variation on sclerite form as a possible defense response against a simulated predator present in the deep habitat, [11,13]. Transplant experiments of Porites lobata morphotypes showed signals of adaptive phenotypic plasticity for skeletal characteristics [14] and a strong genotype influence for stress resistance traced with biomarkers [15]. Shallow and deep morphotypes of Eunicea flexuosa, a Caribbean gorgonian, exhibited low phenotypic plasticity of sclerites and a strong genetic divergence signal [16]. Ow & Todd (2010) described a clear adaptive plasticity response of Goniastrea pectinata in a depth cline, which was supported modeling it against light irradiance [17]. Finally, Bongaerts et al. (2011) detected adaptive plasticity for light conditions in Seriatopora hystrix involving a situation of adaptive divergence along the depth gradient [18].

The feather-like gorgonian coral Antillogorgia bipinnata (Verrill 1864) together with other groups of Caribbean octocorals, attain broad environmental preferences and sympatric distribution ranges, comprise cases of species complex with marked plasticity undergoing probable incipient ecological speciation processes [19,20,21]. A. bipinnata is distributed along coral reefs from 1 to 45m of depth in Panama, Belize, Bahamas, Florida, Colombia, and absent in the Eastern side of the Western Atlantic. In this bathymetric gradient the species exhibit phenotypic variation on traits like size, coloration, sclerite form and branching pattern. Three basic morphotypes can be recognized over a depth cline, the ‘deep morphotype’, ‘typical morphotype’ and ‘bushy morphotype’ or ‘kallos’ [21], the latter (A. kallos

Bayer) described as a distinct species [22,23]. In some coral reefs, such as Panama, in just 7 m of depth, all 3 morphotypes can be present when there is an abrupt change of depth and reef slope, suggesting that this physiological challenge generates an adaptive morphological response. Remarkably, the clade A. bipinnata-kallos follows a phylogeographic pattern, where morphotypes are polyphyletic per location, suggesting that A. bipinnata and A. kallos actually belong to a same interbreeding species but exhibiting notable plasticity [21].

To identify the contribution of plasticity and genotype in the variability of modular characters for A. bipinnata, a reciprocal transplant experiment was carried out between the

‘deep’ and ‘bushy’ morphotypes from two locations in Bocas del Toro, Panama. With this research we test the degree of plasticity of some modular traits related to bathymetric adaptation in the morphotypes of A. bipinnata, to understand the variability pattern of colonial architecture between morphotypes and to detect the genotype and environmental interactions involved in the colonial phenotypes. In addition, we examined the spawning patterns of the different morphotypes at the same sites, where the experiment took place.

The information derived from the reciprocal transplant experiment and spawning documentation will provide key elements to understand the phenotype divergence and philopatric signals present in the A. bipinnata-kallos complex and will serve as a reference to understand the evolutionary mechanisms and patterns of diversification for a remarkable number of species from this subclass that show marked phenotypic variation related to environmental gradients [23,24].

MATERIALS AND METHODS

Antillogorgia bipinnata and spawning record

Based on molecular and morphological evidence, Antillogorgia bipinnata (Verrill) has been recently reassigned from the genus Pseudopterogorgia [25]. It is a shallow water Caribbean coral with a close association to the photosynthetic endosymbiont Symbiodinium clade B1

[26,27,28]. Like its sister species A. elisabethae, A. bipinnata is a surface brooder with restrictions on larvae dispersion, showing a philopatric pattern and a suspected strong geographic structure of populations [29,30]. Populations of the complex A. bipinnata-kallos are grouped in clusters of reefs over the Southern Caribbean, the Mesoamerican Reef, with

Panama and Belize populations closely related [31], and the Florida-Bahamas region. As a modular organism, with polyps and branches as repetitive unit, A. bipinnata is equipped with a flexibility and versatility of parts conforming the colonial architecture [9]. Modular traits discriminating the bushy and deep A. bipinnata morphotypes were used in the phenotypic plasticity test. Colonies of the ‘deep’ morphotype have a bigger size, with a higher length of principal axis and secondary branches, and higher internodal distances, but a lower number of secondary branches in contrast to the ‘bushy’ morphotype. These differences in the architectonical pattern between morphotypes represent a variation in the distribution and density of polyps across the colony. It has a feasible role in nutrient capture, overall photosynthetic rate and physical stress that can be directly related to adaptive responses to environmental variables in the depth cline. The A. bipinnata gametes are released and fertilized near to the new moon during the months of October-December (personal observations). With the goal of assessing the timing of spawning between morphotypes, daily observations were made during November-December, 2012 in the localities of the transplant experiment tracking the times and depths of spawn presence.

Study Area

The experimental localities, Hospital Point and Crawl Key, Bocas del Toro, are at the north part of the Panamanian Caribbean, in the vicinity of the Smithsonian Tropical Research

Institute (STRI) Station, (Fig. 1A). These locations exhibit some environmental differences.

Hospital Point is a protected reef with abundant suspended particles and low water-motion compared to Crawl Key, 15km distant, which is an exposed reef flat, characterized by eroded coral skeletons and higher light penetration. In shallow habitats, waves tend to be more constant and higher than the deep habitats in both sites. Shallow habitats are characterized by coarser sand with corals established over rocks and hard structures. In deep habitats there is a short slope with higher sediment perturbation. Data for temperature and illuminance (total luminous flux per unit area) were recovered in the transplant establishment with HOBO temperature and light data loggers (Hobo Water Temp

Pro, Onset Computer Corp., Bourne, Mass). In the shallow habitats of the two localities, temperature ranged between 26-310C in comparison with 26-300C for deep habitats.

Illuminance had a higher variation with the shallow habitats of Crawl key ranged between 0 and 35800 lux and 0 and 30300 lux for Hospital Point. For deep habitats, 0 and 14400 lux for Crawl key and between 0 and 2900 lux for the Hospital Point.

Establishing reciprocal transplants and data collection To assess the response of modular traits after transplantation, we took advantage of the phenomenon of overcompensation in A. bipinnata, a fast apical response of branching after injury [32]. Fragments of approximately 30cm were sectioned from 32 healthy colonies of the bushy and deep morphotypes in the localities of Crawl Key and Hospital Point, near the

STRI facilities in Bocas del Toro, Panama. The 16 colonies collected from each locality were at least 12m apart from each other, a total of 21 bushy and 11 deep morphotypes. Each colony was settled in duplicate for shallow (2-3 meters) and deep (7-8.5 m) habitats of the two locations (256 pieces), i.e. one control (kept in the native habitat) and three experimental units (Fig. 1B). With colony segments in addition to different colonies from the same morphotype used in the transplantation, we combined genotypic and individual replicates maximizing the estimation power of plastic patterns [33]. To hold the explants steady in position, each one was fixed to a piece of pvc-pipe by pvc-clamps [32]. Pictures were taken (PowerShot G12, Canon®) after the establishment of transplants, during July

13-14, 2011, and at the end of the experiment, in October 17, 2011, 13 weeks later. A background grid (white acrylic board) was used as metric reference for image correction and digital measurement.

Fig. 1. Locations and design of the reciprocal transplant experiment. (A) Map of Bocas del Toro, Panama, showing the localities of Hospital point (black star) and Crawl key (red circle). (B) Schematic representation of experiment with rows signaling the controls and the direction of transplants between habitats and localities.

Digital processing and statistical analysis.

The Photoshop® software was used for the digital image processing, setting pictures to a single optical plane, and ImageJ® for measuring the traits by transforming the scale from pixels to metric units [34]. Modular traits recorded were the vertical growth rate (1) by measuring the length of the new main axis generated; the horizontal growth rate (2) by randomly selecting at least 10 secondary branches, measuring the length at the end of the experiment and subtracting it from the initial one; the number of new branches generated after injury (3), in the growing apical segment as well as branches from old secondary branches and finally, the internodal distances of these new secondary and tertiary branches

(4) generated after the 13 weeks of the experiment.

To explain the variance in the traits measured between morphotypes and sites of destiny, a GLM test (Generalized Linear Model) was applied taking into account the effect of

the independent variables separately and jointly affecting the trait performances.

Here, we defined as independent variables the morphotype (2), referring to colonies

grouped by bushy or deep; and target habitat (2), referring to the shallow or deep

sites where segments were transplanted. Due the differences between localities, an

additional test was applied separating target habitat in the four destinations (4).

Additionally as fixed effect, the interaction between morphotype x target habitat was

assessed on traits performance. Prior to this, normality and homogeneity in the data

were considered by a Levene test. In turn, due to the different number of replicates

between treatments, a Bonferroni correction was performed, and a p value <0.05

used. Using the median values (M) for each of the four traits, a statistical significant

morphotype effect indicates that genotype differences may explain the response. A

significant target habitat effect over the trait variation indicates plasticity, it means

reliable variation in the trait correlated with environment. A significant morphotype

by target habitat interaction indicates a genotype by environmen effect on the

response (G X E), where a variation in the degree of plasticity between morphs is

detected. Here, a significant morphotype x target habitat effect indicates that

morphotypes express different plastic responses to the depth cline. All analyses

were performed using SPSS® software, version 20 (IBM).

RESULTS

Low mortality occurred in the 256 explanted segments along the 13 weeks of the reciprocal transplant experiment. At the end of the experiment just 15 segments died; defining it as dead when there was considerable loss of tissue (> 50%). Eleven of these segments belonged to the deep Crawl Key destination. Sixty additional segments were lost in the photo analysis due to doubtful code identification or unsatisfactory photo recovery of traits assessed; i.e. that was not possible to identify in the picture the code of the colony, the metric references or it was not possible to visualize correctly some of the traits. No predation or diseases were detected on the remaining 92,3% explants, providing big enough sample size to the assessment of the fixed factors.

The GLM test for each of the four traits considered in the morphotypes of Antillogorgia bipinnata indicates a pattern of plasticity but also a genetic component explaining the variance after 13 weeks of transplantation (Table 1). Using the median values (M) to graph the reaction norms, vertical growth rate response (Fig. 2) show that higher values were common at shallow habitats, particularly high at shallow natives from Hospital Point.

Except the Hospital Point deep segments transplanted to Crawl Key shallow habitat, the slopes between these habitats were in the same direction and were not parallel. Stronger differential slopes were found for transplants between localities. Against the positive values for vertical growth rate, in most cases, horizontal growth rates were more variable and reached negative values, indicating losts of secondary branch tissue (Fig. 3). Higher horizontal growth rates values were noticeable in shallow habitats, with peaks in shallow natives from Hospital Point (M=2.77, SD=1.53) compared to deep natives from the Crawl

Key locality (M=-6.35, SD=2.73). Most of slopes were in the same direction and nearly parallel. GLM analysis yielded a significance variation (p<0.001) for vertical growth rate, indicating that at least one of the destinations was affecting the main axis growing of morphotypes but that an interaction with the variable genotype is explaining the variance.

Table 1. Results of General Lineal Model of each trait assessed for Morphotype, Habitat and

Morphotype X Habitat interaction variables. Fisher (F) and Probability (P) values are showed, asterisks indicates significant p values (P<0.05)

Morphotype Habitat Morphotype X Habitat Trait F P F P F P Vertical growth rate 3.84 0.058 1.177 0.286 113.21 <0.001* Horizontal growth 1.22 0.276 1.365 0.251 0.621 0.436 rate New branches 1.63 0.210 6.692 0.014* 8.809 0.006* Internodal distances 0.61 0.440 0.255 0.617 12.339 0.001*

hhhh HPs CKs HPd CKd

HPs CKs 20 HPd CKd CK 18

16 HP HP 14

12 HP Native HP 10 HP HP Native

8 CK Native CK Native 6 CK CK 4 HP

Vertical Growth Rate Growth Vertical CK CK 2 HP CK

−2 S S D D

Fig. 2. Reaction norms for vertical growth rate of A. bipinnata. In the X-axis are the different habitats

S=shallow, D=deep. In the Y-axis is the vertical growth rate in mm. Black and red dots represent the median magnitudes of the main axes growth rate and error bars the standard deviation. HPs= Hospital Point shallow,

HPd= Hospital Point deep, CKs= Crawl Key shallow, CKd= Crawl Key deep.

hhhh HPs CKs HPd CKd

4 HPs CKs HPd CKd 3 HP Native CK Native 2

1 HP

0 CK −1 CK CK −2 CK −3 CK HP −4 HP CK −5 HP HP −6

−7 HP Horizontal Growth Rate Growth Horizontal CK Native −8 HP Native

−9

S S D D

Fig. 3. Reaction norms for horizontal grow rate of A. bipinnata. In the X-axis are the different habitats s=shallow, d=deep. In the Y-axis is the vertical growth rate in mm. Black and red dots represent the median magnitudes of secondary branches growth rate and error bars the standard deviation. HPs= Hospital Point shallow, HPd= Hospital Point deep, CKs= Crawl Key shallow, CKd= Crawl Key deep.

Growth of new branches (Fig. 4) was higher in shallow habitats compared to the deep ones.

A large number of branches were generated in the explants from Crawl Key deep to Crawl

Key shallow (M =33.75, SD=27.72) and to Hospital point shallow (M= 15.75, SD=14,75). Due to these two high values in comparison to the deep natives of Crawl Key (M= 0 SD=1.16) and Hospital point (M= 0 SD=0.16), and even the shallow natives, Figure 4 shows separately the reaction norms for each kind of transplant, looking for a better visual inspection. A nonparallel pattern of branch promotion is evident, where the branch promotion of Hospital

Point deep explants are fairly lower than Crawl Key deep in shallow habitats. In the case of internodal length, the GLM test was performed only in the case of Crawl Key deep morphotypes with new branches in the shallow habitats. This is because of the dependence of new branches to perform internodal distance measurements, resulting to non-significant samples sizes in other transplants. Even with this restriction, the level of confidence for a genotype x environment was highly significant (p=0.006), and the reaction norm (Fig. 5) showed a reduced internodal distance between new branches. These reaction norm directions resembled the native ones of the shallow habitats, where bushy morphotype presents a higher density of secondary branches in a shorter main axis, evidenced by the lower values of internodal distances for Crawl Key natives (M =1.6, SD=0.12) and Hospital

Point natives (M =1.77, SD=0.18).

20 Native HPs Native CKd Native CKs Native CKd

NumberNewof branches 0

5 Native HPs Native CKs Native HPs Native HPd

NumberNewof branches −5

Native CKs 2 Native HPd Native CKd Native HPd

−2 NumberNewof branches

Fig. 4. Reaction norms for new branches promotion of A. bipinnata. A high number of new branches were found in Crawl Key morphotypes in shallow habitats. For a better clarity, transplant experiments are shown separately (y-axis split). Black and red dots represent the median magnitudes and error bars the standard deviation. HPs= Hospital Point shallow, HPd= Hospital Point deep, CKs= Crawl Key shallow, CKd= Crawl Key deep.

3.0

2.8

2.6 HPs Native CKd 2.4

2.2 CKs

2.0

Native HPs 1.8 Internodal length (mm) length Internodal

Native CKs 1.6

1.4

Fig. 5. Reaction norm for internodal length of Crawl Key deep transplant in shallow habitats of A. bipinnata. In Y-axis the internodal length in mm. Black and red dots represent the median magnitudes and error bars the standard deviation. HPs= Hospital Point shallow, CKs= Crawl Key shallow, CKd= Crawl Key deep.

Finally, the monitoring over habitats and localities during 2012 showed revealing patterns of female spawning (Fig. 6). On November 21st, eight days after the new moon of November

13, shallow morphotypes from Crawl Key were the first releasing eggs to the surface. Daily records revealed that A. bipinnata spawned progressively every 2-3 m in depth range, with the first eggs released from the deep female colonies at 8 m on November 25, at which point spawning had completely ended in the shallow habitat, and finishing 3 days later. In the

Hospital Point locality, surface brooding was first noticeable on November 27 in deep colonies at 8m, followed by a fast spawn towards shallower colonies starting at 3 m depth on November 30 and finishing three days later.

Fig. 6. Colonies of A. bipinnata with surface brooding. Colonies from Crawl Key locality during spawning on

November, 2012. (a) Freshly released eggs (November 21) at 3 m depth; (b) Five days larvae (November 22) at 2.5 m depth; (c) detail of five-days larvae November 23, 2.5 m depth; (d) Freshly released eggs, November

25, 2012, 6 m depth. 121x97mm (300 x 300 DPI).

DISCUSSION

The GLM tests and reaction norms graphs for each of the traits pointed to adaptive phenotypic plasticity with a genetic component in a classical genotype by environment model of phenotypic response. The elevated survival rate of foreign transplants and the directions towards native values of reaction norms indicated some grade of adaptive response. The general trend of variation in the four traits tested resembled the native transplants performance. In the case of bushy segments transplanted into deep habitats, a lower rate of vertical and horizontal growth, lower new branches and higher internode distances were found, opposing to the general response of foreign deep segments into shallow habitats of the two localities. Even when the standard deviations were high, the tendency in the way the four traits varied, highlights a grade of phenotypic plasticity in an adaptive fashion (i.e., slopes follows the same direction). The vertical and the horizontal growth rates of all transplants were higher in the shallow habitats, which is counterintuitive having in mind that shorter main and secondary branches characterize bushy morphotypes. Probably, these growth rates could be explained by an initial difference in response against habitat after injury and insufficient time elapsed to display a reversion in growth rate behaviors.

The slopes of reaction norms inside localities are almost parallel, but between localities, nonparallel patterns were found. For all the traits evaluated in the shallow-deep transplants inside localities, the responses were similar but not the same, which points to a genetic component involved in the different responses [9]. The number of new branches clearly illustrates this observation with the deep morphotype from Crawl Key having high values of new branches in shallow habitats, against the almost null branch promotion of bushy morphotypes from the two localities in the Crawl Key deep habitat. All the kinds of reaction norms corresponded to a genotype x environment interactions [35], in our case more specifically a morphotype x environment interaction.

A pattern of maladaptive reaction was just oberved for some transplants in the vertical growth rate. Here, the deep morphotype from Hospital Point in the shallow habitat of Crawl

Key, and the bushy morphotype of Crawl Key in deep habitats, had less optimal performance than expected without exhibiting plastic response. Thus, in addition to the differences in native performances of the same morphotype from the two localities, a stronger G X E interaction was taking place, involving a lower adaptive plasticity and a higher ecological divergence due to environmental differences between Hospital Point and

Crawl Key [36]. It will be desirable in a future assessment, to get a local adaptation measure for A. bipinnata morphotypes in these localities. A longer term reciprocal transplant is advisable, where life history attributes such as gametogenesis, number of reproductive cycles/year and larval production would represent better proxies for fitness than branch growth, even when this could be highly challenge for corals. With that kind of information it may be possible to test if there is a significant reduction of fitness of the bushy morphotypes from Crawl Key in the deep Hospital point habitat, representing a possible case of gene flow reduction promoted by a maladaptive plastic response to the environment

[33].

Research on spawning is constrained by the very limited amount of time per year available for study, but testing the synchrony of the process could be a step towards identifying fertilization barriers [37]. Personal observations during 2001, 2003 and 2011 had shown that although the beginning of the surface brooding in A. bipinnata is not quite predictable in terms of lunar phases, sites and colonies, the event seemed to differ consistently with depth. This has been supported with the asynchronic surface brooding during November 2012. Although between localities the shallow to deep order of spawn was not the same, it is a remarkable observation the spawn synchrony over the colonies inside habitats, and the asynchrony between the shallow and deep morphotypes at different sites. Pheromonal stimuli might be contributing in the synchrony of neighboring colonies from the same morphotype, and at the same manner the separation in time between morphotypes distributed in the depth gradient [37]. Differences in the brooding time between morphotypes can be related to the asynchronous time of gamete maturation. A case of temporal reproductive mismatch has the capacity to reduce population connectivity and could promote rapid evolutionary processes [38,39]. Even when we must be cautious not to over-interpret the observed results, brooder gorgonian corals are markedly philopatric, where larvae usually settles close to their mother colonies [40]. Then, a scenario of prezygotic isolation producing assortative mating could be plausible, in which depth- associated spawning can speed up the selection process for particular genotypes, a testable mechanism contributing to a scenario of ecological speciation. If a process of genetic phenotype fixation, where a rapid evolution will lead to divergence between subpopulations, is the case on the G X E interactions evidenced in the bushy and deep morphotypes of A. bipinnata, a molecular evaluation of adaptive genetic divergence, coupled with a functional identification of genes with signals of positive selection, could provide an integral perspective to test the current hypothesis of an incipient ecological speciation mediated by phenotypic plasticity mechanisms.

ACKNOWLEDGMENTS

The Smithsonian Tropical Research Institute (STRI) (Senior Latin American Fellow to J.A.

Sánchez) and Universidad de los Andes (Facultad de Ciencias), Colombia, sponsored this research. Comments and informal discussion with Howard R. Lasker, Harilaos Lessios and

Carlos Prada are greatly appreciated. Thanks to Oscar Ramos and Daniel Galindo for statistical support and advises. Thanks to Luisa Duenas Adriana Sarmiento, Diana

Ballesteros, Rocio Acuna and Cindy Gonzalez for field assistance. Comments from Nick

Schizas and Zaira Garavito greatly improved the manuscript.

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