Royal Botanic Gardens, Kew UK Overseas Territories

Interim report for the project "Population status and gene bank development for the endangered species Varronia bellonis" [F15AC01225], 01 August 2017 to 31 July 2018

Figure 1. San Juan WSR-88D radar image of Hurricane Maria at 0950 UTC 20 September, just before landfall in Puerto Rico, showing the more dominant outer eyewall. This was the last image from the radar before it was destroyed. Source: (Pasch, Penny and Berg, 2018).

Prepared by: Dr Martin A. Hamilton, Research Leader (UKOTs), UK & Islands Programme, Conservation Science Department, Royal Botanic Gardens, Kew

Hamilton, M.A. (2018). Interim report for the project "Population status and gene bank development for the endangered species Varronia bellonis" [F15AC01225], 01 August 2017 to 31 July 2018

Acknowledgements The project team wish to thank the funder of the activities report herein: The USFWS through the CFDA Program 15.657 1 Besides those individuals listed under ‘Project participants’, the team would also like to thank Amneris Siaca, José Salguero, Gustavo Olivieri, Russell Smith and Maria Chubb for their assistance and support.

Suggested citation Hamilton, M.A., 2018. Interim report for the project "Population status and gene bank development for the endangered species Varronia bellonis" [F15AC01225], 01 August 2017 to 31 July 2018. Richmond, Surrey, U.K.: Royal Botanic Gardens, Kew. This work is licensed under a Creative Commons Attribution-NonCommercialNoDerivatives 4.0 International License. All images copyright ©Dr M.A. Hamilton unless otherwise noted. Please direct queries and feedback to: Dr Martin A. Hamilton Research Leader (UKOTs) Conservation Science Department The Herbarium, Kew Richmond, Surrey TW9 3AE United Kingdom +44 (0)2083325020 +44 (0)2083325278 [fax] [email protected]

This document has been produced with the financial assistance of the US Fish and Wildlife Service funded project [F15AC01225] “Population status and gene bank development for the endangered species Varronia bellonis” administered by Puerto Rico’s Department of Natural and Environmental Resources. 1

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Contents

Acknowledgements................................................................................................................................. 2 Suggested citation................................................................................................................................... 2 Contents .................................................................................................................................................. 3 Figures ..................................................................................................................................................... 3 Project participants ................................................................................................................................. 4 Acronyms ................................................................................................................................................ 5 Introduction ............................................................................................................................................ 6 Images and supplementary data ........................................................................................................ 6 Project Outcome Statement ............................................................................................................... 7 Project Objectives ............................................................................................................................... 7 Year three (01 August 2017 to 31 July 2018) project activities .............................................................. 7 Project publicity .................................................................................................................................. 8 Project performance review ............................................................................................................... 9 Next steps and key recommendations ................................................................................................. 10 References ............................................................................................................................................ 10 Kew Financial Report ............................................................................................................................ 11

Figures Figure 1. San Juan WSR-88D radar image of Hurricane Maria at 0950 UTC 20 September, just before landfall in Puerto Rico, showing the more dominant outer eyewall. This was the last image from the radar before it was destroyed. Source: (Pasch, Penny and Berg, 2018)................................................. 1 Figure 2: Summary of Varronia bellonis collections and observations made between 01 April 2016 and 31 July 2018. ........................................................................................................................................... 8 Figure 3. Current known distribution of Varronia bellonis derived from field survey between April 2016 and July 2018. ......................................................................................................................................... 8

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Project participants 2 Initials BR BS CC CM DRR EA EM ES ILR JCB JM JPB JRC JS JV MAH MDS MG MLF MR NJ OM RMM RPS SB TMH

Name Brian Ramos Barbara Sanchez Dr Colin Clubbe Dr Chris Malumphy Damaris Roman Ruiz Edwin Avila Edwin Muniz Dr Eugenio Santiago Iván Llerandi-Román José Cruz-Burgos José Martinez Jong Piel Banchs Plaza Jesus M. Rios Cruz Jose Sustache Jeanine Velez Dr Martin Hamilton Dr Michele Sanchez Dr Miguel A. Garcia Marisel Lopez Flores Marelisa Rivera Dr Nilda M. Jimenez Marrero Omar Monsegur Roseanne Medina Miranda Rafael Picó Seda Sara Barrios Tom Heller

Organizational affiliation DNER Botany Lab Technician, MAPR Head of Conservation Science, Kew Fera Science Parrot Project, DNER Maricao Forest Manager, DNER USFWS Professor and UPR Herbarium Manager USFWS USFWS USFWS Parrot Project, DNER Parrot Project, USFWS Botanist and SJ Herbarium Manager, DNER MAPR manager, University of Puerto Rico, Mayaguez Research Leader (UKOTs), Kew Caribbean TIPAs Programme Coordinator, Kew CLCC, USFWS Puerto Rican Parrot Recovery Program Project Leader, USFWS USFWS DNER Biologist/Botanist, USFWS Parrot Project, USFWS Sub-secretary, DNER Islands Conservation Partnership Co-Coordinator, Kew Islands Conservation Partnership Co-Coordinator, Kew

Many individuals have been involved in the project over the past year. Without the support of the people listed here, this work would have not been possible.

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Acronyms Acronym DNER GIS GPS Kew MAPR MSBP PR SJ UK UKOTs

Definition Department of Natural and Environmental Resources Geographic information system Global positioning system Royal Botanic Gardens, Kew Herbarium of the University of Puerto Rico at Mayagüez Millennium Seed Bank Partnership, RBG, Kew Puerto Rico Herbarium of the Department of Natural and Environmental Resources United Kingdom of Great Britain and Northern Ireland United Kingdom Overseas Territories

UPR

Herbarium of the University of Puerto Rico Botanic Garden

UPRRP

Herbarium of the University of Puerto Rico at Rio Piedras

US USDA USFS USFWS

United States of America US Department of Agriculture US Forest Service US Fish and Wildlife Service

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Introduction This project is part of on-going regional collaborations with several Puerto Rican collaborators, namely the US Fish and Wildlife Service (USFWS) Caribbean Ecological Services Field Office, University of Puerto Rico (UPR) Mayagüez Department of Biology Herbarium (MAPR) and the Department of Natural and Environmental Resources (DNER). The Royal Botanic Gardens, Kew (Kew) submitted a proposal to the USFWS Southeast Region Caribbean Ecological Services Field Office (CESFO) in Boquerón, Puerto Rico to assist in the recovery efforts related to the federally listed, endangered plant species Varronia bellonis. This report details the activities undertaken by Kew from 01 August 2017 to 31 July 2018 in collaboration with Puerto Rican colleagues under the USFWS funded project [F15AC01225] “Population status and gene bank development for the endangered species Varronia bellonis” administered by DNER. Survey and monitoring activities undertaken as part of a synergistic project are detailed in the report prepared by the author and listed below 3. Hamilton, M.A., 2018. Interim report for the project "Biogeography and reproductive biology of the endangered species Varronia bellonis" [F17AC01111], 01 September 2017 to 31 July 2018. Richmond, Surrey, U.K.: Royal Botanic Gardens, Kew.

Images and supplementary data Images and supplementary data have been uploaded to a file sharing website and can be accessed here.

See: https://www.researchgate.net/project/Varronia-bellonis-Boraginaceae-Conserving-an-endemic-PuertoRican-plant 3

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Project Outcome Statement The endangered shrub, Varronia bellonis, will be secured in at least two new gene bank collections and the meta-population status will be evaluated by international experts. Population genetic analyses will be undertaken to determine within and between population diversity. Consolidated regional and international partnerships will enable sound and scientifically based recovery actions to be undertaken to secure Varronia bellonis into the future.

Project Objectives 1. To update the global distribution of the species through systematic surveys within suitable habitat including non-traditional sites using GIS; 2. To develop gene bank collections (i.e. seed, living plants, DNA samples) in at least two new locations (e.g. UPR-RUM and Kew); 3. To explore the genetic variation within and among populations using existing microsatellites developed for Varronia rupicola; 4. To explore the effects of the species reproductive biology (dioecious breeding system) on the patterns of genetic variation as well as considering the implications for the species conservation; and 5. To opportunistically address questions related to the species reproductive biology (e.g. seed dispersal and pollination).

Year three (01 August 2017 to 31 July 2018) project activities There were no survey or monitoring activities specifically undertaken as part of this project. The only activities in the reporting period were administrative (e.g. grant extension request, modification of project timeline, project steering group meetings, project reporting, data compilation). Due to the impacts of Hurricane Maria in September 2017, a formal request for an extension of the project was made to the USFWS CESFO for agreement F15AC01225. The extension was specifically requested since the Puerto Rican student, Anthony Maldonado, that will undertake the genetic analyses as part of a joint Royal Botanic Gardens, Kew and Queen Mary University MSc project was unable to start his course in October 2017 due to Hurricane Maria. The student was guaranteed a place on the same course starting in October 2018 and was, therefore, not able to undertake the genetic analyses originally planned for May/June 2018. For this reason, a one-year extension of the project was granted to accommodate the delay of the student’s course and project due to the impacts of the hurricane. The original period of project performance of 31st December 2018 was formally extended through 31st December 2019 to enable the Puerto Rican student to undertake the analyses in May/June 2019. Field based activities have been undertaken in the three historic locations known to have supported populations of Varronia bellonis and extant populations have been confirmed in and around the Maricao, Rio Abajo and Susúa State Forests. The project team have documented several new individuals and sub-populations, observing 299 mature plants, 57 immature plants or saplings and 32 seedlings. Recruitment in Rio Abajo and Susúa appears to be very poor. 7

Hamilton, M.A. (2018). Interim report for the project "Population status and gene bank development for the endangered species Varronia bellonis" [F15AC01225], 01 August 2017 to 31 July 2018 To date, samples from 160 wild and 4 cultivated Varronia bellonis plants have been collected for a population genetics study. At least one of the samples from Rio Abajo is thought to be a reintroduced plant from the state nursery following the Highway 10 development. No other reintroduced plants are known to be extant. Collecting area Maricao Río Abajo, Utuado, Ciales Susúa

# Recorded points 250 42 1 293

# DNA samples # Mature plants # Saplings # Seedlings 127 257 46 29 32 41 11 3 1 1 0 0 160 299 57 32

Figure 2: Summary of Varronia bellonis collections and observations made between 01 April 2016 and 31 July 2018.

Figure 3. Current known distribution of Varronia bellonis derived from field survey between April 2016 and July 2018.

Project publicity Publicity for the project was generated through Twitter (@DocMartinKew, #KewPR) along with a dedicated project page on ResearchGate.

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Project performance review In the third year, the project was effectively put on hold due to the impacts of Hurricane Maria. The storm resulted in the genetic study being postponed for one year and a formal extension of the project approved through 31st December 2019. A synergistic project, "Biogeography and reproductive biology of the endangered species Varronia bellonis" [F17AC01111], continued collecting baseline data that is providing information toward the objectives of this project. The close collaboration between Kew, DNER and CESFO staff specifically addressed the project objectives as described below. 1. To update the global distribution of the species through systematic surveys within suitable habitat including non-traditional sites using GIS; Review: Surveys undertaken in all areas with historic records for the species (e.g. in and around Maricao, Rio Abajo and Susúa State Forests) using project developed GIS. 2. To develop gene bank collections (i.e. seed, living plants, DNA samples) in at least two new locations (e.g. UPR-RUM and Kew); Review: DNA samples and seeds have been collected during surveys since April 2016 for developing gene bank collections. All collections are currently held at UPR-RUM and SJ herbaria. 3. To explore the genetic variation within and among populations using existing microsatellites developed for Varronia rupicola; Review: Activities not possible due to impacts of Hurricane Maria. 4. To explore the effects of the species reproductive biology (dioecious breeding system) on the patterns of genetic variation as well as considering the implications for the species conservation; Review: Three years of observations undertaken, and hypotheses formed during field surveys and literature review. Synergistic project developed and funded to explore this topic in more detail. 5. To opportunistically address questions related to the species reproductive biology (e.g. seed dispersal and pollination). Review: Camera trapping undertaken in Maricao during December 2016 and December 2017. Synergistic project developed and funded to explore this topic in more detail.

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Next steps and key recommendations In year four, the author will supervise an MSc student from Puerto Rico to undertake the proposed population genetic study to inform the species recovery and deliver the final project output. This is planned to be undertaken in the UK, but it is reliant on securing export permits. The project steering group have discussed this matter at length and the application process is underway. If an issue is encountered, a university lab in Puerto Rico/USA will be approached for assistance. Ten microsatellite primer pairs selected for Varronia rupicola will be used to test their usefulness for detecting populations in Varronia bellonis. Available historic herbarium specimens will also be sampled to test for a loss of genetic diversity. Depending on the results of the initial genetic testing of the existing microsatellites, it may be necessary to develop bespoke markers, or ideally, undertake another method like RAD-Seq as detailed in the original proposal. This would require further funding to be secured.

References Pasch, R. J., Penny, A. B. and Berg, R. (2018) National Hurricane Center Tropical Cyclone Report Hurricane Maria (AL152017) 16-30 September 2017. Miami, Florida. Available at: https://www.nhc.noaa.gov/data/tcr/AL152017_Maria.pdf.

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Kew Financial Report There was no financial expenditure during this reporting period. Project finances remain as reported in the year two project report (see below) with the only expenditure for the project under the budget line “Travel and Subsistence”. Copies of all receipts and financial claims against the project budget are held in digital format on Kew’s financial management system under project code 11216-100. Expenditure under the budget line “Lab work” will commence in year four once the student’s MSc project officially starts. Table 1. Project expenditure as of 31st July 2018.

Total budget USD

$ 20,000.00 Lab work budget USD

$ 12,285.00

Total expense USD

$ 8,231.99

Total variance USD

$ 11,768.01

T&S budget USD T&S expense USD T&S variance USD Expense description USD

$ 7,715.00 $ 8,231.99 $ -516.99 Amount

DNA extractions $5/sample (total of 672 samples) PCR $75/96 samples (total of 672 samples) 10 existing microsat primers $1200/96 samples (total of 672 samples) Lab work expense USD Lab work variance USD Expense description USD

Varronia bellonis project Expenses JULY 2016 USD

$ 3,234.78

~

$

-

Varronia bellonis project Expenses OCT 2016 USD

$ 1,460.21

~

$

-

Varronia bellonis project Expenses MAR 2017 USD

$ 3,537.00

~

$

-

$

3,360.00

$

525.00

$ 8,400.00 $ $ 12,285.00 Amount

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